CyCAT half-body molecules comprising sterically hindered moieties

Abstract

The present disclosure provides CyCAT half-body molecules and complementary pairs of such half-body molecules capable of associating with each other on the surface of a cell expressing two target antigens of interest. The tri-specific heterodimeric antibody molecules thus formed on the cells and on the target are able to engage and stimulate cytotoxic T cells to disrupt tumor cells. The disclosure further provides half body molecules having sterically hindered moieties attached thereto that reduce heterodimer formation of such half bodies in the presence of only one target antigen of interest or in the absence of two target antigens of interest.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to CyCAT molecules and complementary pairs of corresponding CyCAT half molecules that make up these CyCAT molecules, that are similar and activated at the surface of target cells. The resulting trispecific heteromeric antibody molecules formed on the cell and on the target are able to engage and stimulate cytotoxic T cells to destroy tumor cells via the reconstituted aCD3-Fv antibody fragment. The present disclosure also provides strategies to further optimize the safety profile of such CyCAT directed therapies by attaching a steric hindrance moiety (also referred to herein as a "Guard-Domain") to at least one of the half molecules of a complementary pair of half molecules. This combined approach is particularly useful to minimize toxic effects associated with antibody-based therapies involving the recruitment of immune cells to destroy cancer cells. BACKGROUND

[0002] Therapeutic concepts based on the use of multispecific antibodies generally rely on the simultaneous binding to cell surface antigens present on cancer cells and cell surface antigens present on immune effector cells with the aim to activate immune effector cells only in the vicinity of tumor cells (Miller and Kontermann, Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010, 24(2): 89-98). One of the antigens that has been most intensively investigated in this context is CD3, a well-established antigen that stimulates T cells with important therapeutic implications. In this therapeutic concept, cancer cells and cytotoxic T cells are bridged via bispecific antibodies employing CD3 targeting binding domains, which leads to the formation of an immunological synapse between cancer cells and cytotoxic T cells, allowing the T cells to directly kill the cancer cells.

[0003] A significant problem in CD3 targeting therapies is its dose-limiting adverse reactions due to the T cell stimulating capacity inherent to CD3 binding domains, regardless of the presence of target cells, or due to the binding of bispecific antibodies to healthy tissues that can also express the cancer related marker of interest. Strategies have been developed to provide bispecific antibodies in a similar "prodrug" approach, in such a way that the CD3 binding moiety is in an "inactive" state when the bispecific antibody is administered to the patient, but once the bispecific antibody reaches the intended site of action, the CD3 binding moiety is specifically activated.

[0004] WO 2013 / 104804 provides a two-component system comprising a first polypeptide with a first targeting moiety and carrying a VH or VL domain of an aCD3-Fv binding fragment and a second polypeptide with a second targeting moiety and carrying a complementary VL or VH domain of an aCD3-Fv fragment, respectively. Binding of both polypeptides to a cancer cell expressing both target antigens on its cell surface induces dimerization and functional complementation of the CD3 specific variable domains and formation and activation of a trispecific T cell engaging antibody on the cell.

[0005] WO 2015 / 013671 describes a bispecific T cell engaging approach comprising a protease cleavable masking moiety fused to a CD3 binding domain within a conventional bispecific antibody format. Coupling of the masking moiety reduces the ability of the CD3 binding domain to bind to cytotoxic T cells. However, once the bispecific antibody is bound to a cancer cell, the masking moiety is released by the activity of a cancer specific protease in the tumor microenvironment, thereby restoring the ability of the CD3 binding domain to co-engage with T cells.

[0006] WO 2017 / 087789 provides a two-component system similar to the one described in WO 2013 / 104804 but additionally incorporates a domain masking approach in such a way that the masking moiety blocks the interaction and functional complementation of the complementary aCD3 variable domains unless a cancer specific protease in the tumor microenvironment releases the masking moiety. However, the reliance on a tumor specific protease to activate or restore CD3 binding at the desired site of action adds a new layer of complexity to the design of bispecific antibody molecules, e.g. the need to properly incorporate amino acid sequences that can be recognized by such a protease. Furthermore, the actual type, presence and activity of tumor proteases in the tumor microenvironment can strongly depend on the type of cancer and also vary from patient to patient.

[0007] WO 2022 / 051647 discloses a bispecific T cell engaging one-component system that utilizes a similar approach as described in WO 2017 / 087789. Here, two complementary but non-binding antibody variable domains block the formation of an aCD3-scFv present on the same polypeptide unless a cancer specific protease in the tumor microenvironment releases these inert masking moieties.

[0008] However, a common requirement for obtaining functional CD3 binding described in the prior art is the release of one or more masking domains by a tumor specific protease.

[0009] WO 2023 / 006809 discloses various half molecule but differs from the molecules of the present disclosure in that the construct does not encompass a protection domain as described herein.

[0010] Thus, there is a need to improve the development of T cell redirection therapies in this field, by relying on the advantages of using a two-component system as described above, with more optimized safety features in a simpler way as suggested in the art. SUMMARY

[0011] In embodiments, the disclosure provides a pair of half-body molecules comprising

[0012] a) a first half-body molecule (HB1) comprising

[0013] i. a first Fab fragment (Fab1) specific for a first antigen (AG1)

[0014] ii. a first VH (aCD3-VH1) or a first VL (aCD3-VL1) of an Fv fragment (aCD3-Fv) specific for CD3, and

[0015] iii. a first guardian domain (GD1), and

[0016] b) a second half-body molecule (HB2) comprising

[0017] i. a second Fab fragment (Fab2) specific for a second antigen (AG2),

[0018] ii. a complementary VH (aCD3-VH2) or a complementary VL (aCD3-VL2) of the aCD3-Fv, and

[0019] iii. optionally a second guardian domain (GD2),

[0020] wherein HB1 and HB2 are not linked via a covalent bond.

[0021] In embodiments, the disclosure provides a pair of half-body molecules comprising

[0022] a) a first half-body molecule (HB1) comprising

[0023] i. a first antibody or antibody fragment (Ab1) specific for a first antigen (AG1)

[0024] ii. a first VH (aCD3-VH1) or a first VL (aCD3-VL1) of an Fv fragment (aCD3-Fv) specific for CD3, and

[0025] iii. a first guardian domain (GD1), and

[0026] b) a second half-body molecule (HB2) comprising

[0027] i. a second antibody or antibody fragment (Ab2) specific for a second antigen (AG2),

[0028] ii. a complementary VH (aCD3-VH2) or a complementary VL (aCD3-VL2) of the aCD3-Fv, and

[0029] iii. optionally a second guardian domain (GD2),

[0030] wherein HB1 and HB2 are not linked via a covalent bond.

[0031] In embodiments of the disclosure, the pair of half molecules is a pair of complementary half molecules. In embodiments of the disclosure, HB1 and HB2 are capable of forming a heterodimer. In embodiments, HB1 and HB2 are capable of forming a heterodimer with each other. In embodiments, HB1 and HB2 are capable of forming a heterodimer when mixed in solution. In embodiments, HB1 and HB2 are capable of forming a heterodimer on the surface of a cell. In embodiments, HB1 and HB2 are capable of forming a heterodimer on the surface of a cell expressing AG1 and AG2 on its surface.

[0032] In embodiments, the formation of a heterodimer of HB1 and HB2 occurs via dimerization of aCD3-VH1 and aCD3-VL2 or dimerization of aCD3-VL1 and aCD3-VH2. In embodiments, dimerization of aCD3-VH1 and aCD3-VL2 or dimerization of aCD3-VL1 and aCD3-VH2 results in the formation of aCD3-Fv. In embodiments, dimerization of aCD3-VH1 and aCD3-VL2 or dimerization of aCD3-VL1 and aCD3-VH2 results in the formation of a T cell engaging trispecific antibody. In embodiments, heterodimerization of HB1 and HB2 results in the formation of a T cell engaging trispecific antibody.

[0033] In embodiments of the disclosure, GD1 does not substantially bind to aCD3-VH1, aCD3-VL1, or HB1. In embodiments, GD2 does not substantially bind to aCD3-VH2, aCD3-VL2, or HB2. In embodiments, GD1 does not bind to aCD3-VH1, aCD3-VL1, or HB1, and wherein GD2 does not bind to aCD3-VH2 or aCD3-VL2 or HB2.

[0034] In embodiments of the disclosure, each of GD1 and GD2 is a soluble protein, a soluble polypeptide, a soluble globular protein, or a soluble globular polypeptide. In embodiments, GD1 is a soluble protein, a soluble polypeptide, a soluble globular protein, or a soluble globular polypeptide. In embodiments, GD2 is a soluble protein, a soluble polypeptide, a soluble globular protein, or a soluble globular polypeptide.

[0035] In embodiments of the disclosure, each of GD1 and GD2 has a molecular size of less than 70 kDa. In embodiments, GD1 has a molecular size of less than 70 kDa. In embodiments, GD2 has a molecular size of less than 70 kDa.

[0036] In embodiments of the disclosure, each of GD1 and GD2 is selected from the group of albumin, fibrinogen, fibronectin, hemoglobin, transferrin, an immunoglobulin domain, or a fragment thereof. In embodiments, the albumin is human serum albumin or a fragment of human serum albumin. In embodiments of the disclosure, the fragment of human serum albumin is domain 1, domain 2, or domain 3 of human serum albumin.

[0037] In embodiments of the disclosure, the immunoglobulin domain is an IgG CH1, CH2, or CH3 domain or an antibody variable domain. In embodiments, the immunoglobulin domain or antibody variable domain is a VH domain or a VL domain. In embodiments, GD1 and / or GD2 is a VH domain (GD VH ) or a VL domain (GD VL ). In embodiments of the disclosure, GD1 does not comprise the amino acid sequence of an antibody variable domain comprised in aCD3-Fv, Fab1, and / or Fab2.

[0038] In embodiments, the disclosure provides a pair of complementary half-body molecules, wherein if HB1 comprises aCD3-VH1, GD1 can be selected to be a VH domain (GD VH ) but not a VL domain (GD VL ), and wherein if HB1 comprises aCD3-VL1, GD1 can be selected to be a VL domain (GD VL ) but not a VH domain (GD VH ).

[0039] In embodiments, the disclosure provides a pair of complementary half-body molecules, wherein if HB2 comprises aCD3-VH2, GD2 can be selected to be a VH domain (GD VH ) but not a VL domain (GD VL ), and if HB2 comprises aCD3-VL2, GD2 can be selected to be a VL domain (GDVL Instead of the VH domain (GD) VH ).

[0040] In the embodiments disclosed herein, if HB1 contains αCD3-VH1, then GD1 is not GD. VL Furthermore, if HB1 contains αCD3-VL1, then GD1 is not GD. VH In an embodiment of the present invention, if HB2 contains αCD3-VH2, then GD2 is not GD. VL Furthermore, if HB2 contains αCD3-VL2, then GD2 is not GD. VH .

[0041] In the embodiments disclosed herein, GD VH Cannot be with GD VL Forming a functional Fv fragment. In an embodiment, GD VH It cannot form a functional Fv fragment with αCD3-VL1. In the embodiment, GD VH It cannot form a functional Fv fragment with αCD3-VL2. In the embodiments disclosed herein, GD VL It cannot form a functional Fv fragment with αCD3-VH1. In the embodiment, GD VL It cannot form a functional Fv fragment with αCD3-VH2.

[0042] In the embodiments disclosed herein, each of GD1 and GD2 cannot be cleaved from HB1 or HB2 by a tumor-specific protease, respectively. In the embodiments, each of GD1 and GD2 does not contain a cleavage site for a tumor-specific protease.

[0043] In the embodiments disclosed herein, GD1 and αCD3-VH1 or GD1 and αCD3-VL1 are positioned adjacent to each other and parallel on HB1. In the embodiments, GD2 and αCD3-VH2 or GD2 and αCD3-VL2 are positioned adjacent to each other and parallel on HB2.

[0044] In the embodiments disclosed herein, GD1 and αCD3-VH1 or GD1 and αCD3-VL1 of HB1 are present on two different polypeptides. In the embodiments, GD2 and αCD3-VH2 or GD2 and αCD3-VL2 of HB2 are present on two different polypeptides.

[0045] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein the presence of GD1 on HB1 in the absence of AG1 and / or AG2 is...

[0046] i. Inhibit the dimerization of αCD3-VH1 and αCD3-VL2 or the dimerization of αCD3-VL1 and αCD3-VH2, respectively.

[0047] ii. Inhibit the formation of functional αCD3-Fv,

[0048] iii. Inhibit the dimerization of HB1 and HB2, and / or

[0049] iv. Inhibit the formation of T cell-mediated binding trispecific antibody molecules.

[0050] In the embodiments disclosed herein, the inhibition is compared with the inhibition measured in the absence of AG1 and / or AG2 on HB1 and in the absence of GD1 on HB1.

[0051] In embodiments, this disclosure provides a pair of complementary half-molecules as described herein, wherein the presence of GD1 is present in the presence of AG1 and AG2.

[0052] i. Does not inhibit the binding of Fab1 to AG1 or the binding of Fab2 to AG2.

[0053] ii. It does not inhibit the dimerization of αCD3-VH1 and αCD3-VL2 or the dimerization of αCD3-VL1 and αCD3-VH2.

[0054] iii. It does not inhibit the formation of functional αCD3-Fv.

[0055] iv. Does not inhibit the binding of αCD3-Fv to CD3.

[0056] v. Does not inhibit the dimerization of HB1 and HB2.

[0057] vi. Does not inhibit the formation of T cell-mediated binding of trispecific antibody molecules, and / or

[0058] vii. It does not inhibit the activity of trispecific antibody molecules in mediating T cell redirection to kill cells with AG1 and AG2 on their cell surface.

[0059] In the embodiments, this disclosure provides a pair of complementary half-molecules as described herein, wherein the presence of GD1 in the presence of AG1 and AG2 does not inhibit the binding of Ab1 to AG1 or the binding of Ab2 to AG2.

[0060] In the embodiments disclosed herein, the inhibition is compared with the inhibition measured in the presence of AG1 and AG2 but in the absence of GD1.

[0061] In the embodiments, this disclosure provides a pair of complementary hemimolecules as described herein, wherein the activity of the trispecific antibody molecules in mediating T cell redirection to kill cells having AG1 and AG2 on their cell surface is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times that of cells having AG1 or AG2 on their cell surface.

[0062] In the embodiments, the activity was determined in an in vitro T-cell cytotoxicity assay as described herein.

[0063] In embodiments, this disclosure provides a pair of complementary hemimolecules, wherein the trispecific antibody molecules mediate the IC50-mediated T-cell redirection killing of cells expressing AG1 and AG2 on their cell surface. 50 The concentration was measured as IC50 under conditions where cells expressing AG1 and / or AG2 were absent. 50 The concentration is at most one-half, one-third, one-quarter, one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, or at most one-tenth.

[0064] In embodiments, this disclosure provides a pair of complementary hemimolecules, wherein the IC50 measured against hemimolecule-pair-induced T cell-mediated killing of cells expressing AG1 or AG2 on their cell surface, in the presence of GD1 on HB1 or GD2 on HB2, is [not specified in the original text]. 50 The concentration and IC50 were determined by the killing effect of T cells induced by half-body pair on cells expressing AG1 and AG2 on their cell surface. 50 The concentration ratio was increased compared to the same ratio measured for a pair of complementary half-molecules lacking GD1 on HB1 or GD2 on HB2.

[0065] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein IC in the absence of GD1 or GD2 50 (AG1 or AG2) / IC 50 Compared to AG1 and AG2, the presence of GD1 on HB1 or GD2 on HB2 increases IC respectively. 50 (AG1 or AG2) / IC 50 The ratio of (AG1 and AG2), wherein the IC 50 (AG1 or AG2) / IC 50 The ratio of (AG1 and AG2) was defined as the IC50 value determined by the intracellular killing effect (IC50) against half-body-induced T cell-mediated killing of cells expressing AG1 or AG2 on their cell surface. 50The concentration and IC50 were determined by the killing effect of T cells induced by half-body pair on cells expressing AG1 and AG2 on their cell surface. 50 The ratio of concentrations.

[0066] In the embodiments disclosed herein, the IC 50 The concentration ratio increases by at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, 150 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times.

[0067] In the embodiments disclosed herein, HB1, HB2, αCD3-VH1, αCD3-VH2, αCD3-VL1 and / or αCD3-VL2 themselves cannot bind to CD3.

[0068] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein αCD3-VH1 and / or αCD3-VH2 comprise the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 12.

[0069] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein αCD3-VL1 or αCD3-VL2 comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13.

[0070] In embodiments, this disclosure provides a pair of complementary hemimolecules, wherein AG1 and AG2 are the same antigen or wherein AG1 and AG2 are different antigens. In embodiments, AG1 and AG2 are present on the cell surface. In embodiments, AG1 and AG2 are present on the surface of the same cell. In embodiments, the cell is a cancer cell or tumor cell. In embodiments, AG1 and / or AG2 are cancer-associated antigens. In embodiments, AG1 and / or AG2 are expressed on the surface of tumor cells or on the surface of tumor progenitor / precursor cells. In embodiments of this disclosure, the combination of AG1 and AG2 is found only on cancer cells and not on non-cancer cells. In embodiments, the combination of AG1 and AG2 is present on the surface of the same cell.

[0071] In the embodiments disclosed herein, HB1 further comprises a first Fc region, and / or HB2 further comprises a second Fc region. In the embodiments, HB1 further comprises a first Fc region. In the embodiments, HB2 further comprises a second Fc region. In the embodiments, the first Fc region and / or the second Fc region comprises one or more amino acid mutations that reduce the binding affinity of the Fc region to the Fc receptor and / or the Fc region to C1q, and / or reduce its effector function.

[0072] In embodiments, this disclosure provides a pair of complementary half-molecules wherein the first Fc regions of Fab1, αCD3-VH1 or αCD3-VL1, GD1, and HB1 are fused together via peptide linkers. In embodiments, this disclosure provides a pair of complementary half-molecules wherein the second Fc regions of Fab2, αCD3-VH2 or αCD3-VL2, GD2, and HB2 are fused together via peptide linkers.

[0073] In embodiments, this disclosure provides a pair of complementary half-molecules wherein the first Fc regions of Ab1, αCD3-VH1 or αCD3-VL1, GD1, and HB1 are fused together via peptide linkers. In embodiments, this disclosure provides a pair of complementary half-molecules wherein the second Fc regions of Ab2, αCD3-VH2 or αCD3-VL2, GD2, and HB2 are fused together via peptide linkers.

[0074] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein, in the absence of AG1 and / or AG2 and in the presence of GD1 on HB1, the dissociation constant (K) between αCD3-VH1 and αCD3-VL2 or between αCD3-VL1 and αCD3-VH2 is [value missing]. D K was measured in the absence of GD1 on HB1. D At least 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 times.

[0075] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein in the absence of AG1 and / or AG2 and in the presence of GD1 on HB1, the amount of heterodimer formed between HB1 and HB2 is at most one-half, one-third, one-quarter, one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, or at most one-tenth of the amount measured in the absence of AG1 and / or AG2 and in the absence of GD1 on HB1.

[0076] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein, in the presence of AG1 and AG2 and GD1 on HB1, the dissociation constant (K) between αCD3-VH1 on HB1 and αCD3-VL2 on HB2 or between αCD3-VL1 on HB1 and αCD3-VH2 on HB2 is... D The dissociation constant (K) measured in the presence of AG1 and AG2 but without GD1. D They are roughly the same.

[0077] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein, in the presence of AG1 and AG2 and GD1 on HB1, the dissociation constant (K) between αCD3-VH1 on HB1 and αCD3-VL2 on HB2 or between αCD3-VL1 on HB1 and αCD3-VH2 on HB2 is... D () is the K measured in the presence of AG1 and AG2 but in the absence of GD1. D Within twice the amount.

[0078] In an embodiment, this disclosure provides a pair of complementary half-molecules, wherein the amount of heterodimerized HB1 and HB2 in the presence of AG1 and AG2 and the presence of GD1 on HB1 is substantially the same as the amount measured in the presence of AG1 and AG2 but the absence of GD1 on HB1.

[0079] In an embodiment, this disclosure provides a pair of complementary half-molecules, wherein in the presence of AG1 and AG2 and GD1 on HB1, the amount of heterodimerized HB1 and HB2 is within twice the amount measured in the presence of AG1 and AG2 but without GD1 on HB1.

[0080] In embodiments, this disclosure provides a pair of complementary hemimolecules, wherein the activity of the resulting trispecific antibody molecules in mediating T cell redirection killing of cells expressing said AG1 or AG2 on their cell surface in the presence of AG1 or AG2 is at most one-half, one-third, one-quarter, one-fifth, one-sixth, one-seventh, one-eighth, one-ninth, or at most one-tenth of the activity measured in the presence of AG1 or AG2 but in the absence of GD1 on HB1, as determined in an in vitro T cell cytotoxicity assay.

[0081] In the embodiments, this disclosure provides a pair of complementary hemimolecules, wherein the activity of trispecific antibody-mediated T cell redirection killing of cells expressing AG1 and AG2 on their cell surface in the presence of AG1 and AG2 and GD1 on HB1 is substantially the same as the activity measured in the presence of AG1 and AG2 but in the absence of GD1 on HB1, as determined in an in vitro T cell cytotoxicity assay.

[0082] In embodiments, this disclosure provides a pair of complementary hemimolecules, wherein the activity of trispecific antibody-mediated T cell redirection killing of cells expressing AG1 and AG2 on their cell surface in the presence of AG1 and AG2 and in the presence of GD1 on HB1 is up to 5 times the activity measured in the presence of AG1 and AG2 and in the absence of GD1 on HB1, as determined in an in vitro T cell cytotoxicity assay.

[0083] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein the presence of GD1 on HB1 in the absence of AG1 and / or AG2 reduces the ability of αCD3-VH1 on HB1 to dimerize with αCD3-VL2 on HB2 by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the corresponding ability in the absence of GD1 on HB1.

[0084] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein the presence of GD1 on HB1 in the presence of AG1 and AG2 reduces the dimerization ability of αCD3-VH1 on HB1 to αCD3-VL2 on HB2 by no more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or no more than 10% compared to the corresponding dimerization ability in the absence of GD1 on HB1. In embodiments, the dimerization ability is determined in vitro by surface plasmon resonance or biolayer interferometry. In embodiments, the dimerization ability is determined by size exclusion chromatography.

[0085] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein the amount of heterodimer formed between HB1 and HB2 in the presence of AG1 and AG2 is at least 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 times the amount of heterodimer formed in the absence of AG1 and / or AG2.

[0086] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein GD1 and / or GD2 comprise an amino sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0087] In the embodiments, this disclosure provides a pair of complementary hemimolecules, wherein the αCD3-Fv fragment (αCD3-Fv) specifically binds to human CD3.

[0088] In one embodiment, the αCD3-Fv specifically binds to CD3ε. In another embodiment, the CD3 is CD3ε. In yet another embodiment, the αCD3-Fv cross-reactively binds to cynomolgus monkey CD3.

[0089] In the embodiments disclosed herein, the αCD3-VH1 or αCD3-VH2 comprises an HCDR1 region containing an amino sequence of SEQ ID NO: 1 or SEQ ID NO: 9, an HCDR2 region containing an amino sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and an HCDR3 region containing an amino sequence of SEQ ID NO: 3.

[0090] In the embodiments disclosed herein, the αCD3-VL1 or αCD3-VL2 comprises an LCDR1 region containing an amino sequence of SEQ ID NO: 4 or SEQ ID NO: 11, an LCDR2 region containing an amino sequence of SEQ ID NO: 5, and an LCDR3 region containing an amino sequence of SEQ ID NO: 6.

[0091] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB1 is composed of a first polypeptide and a second polypeptide, wherein

[0092] (a) The first polypeptide comprises a heavy chain of Fab1 and αCD3-VH1 or αCD3-VL1 from its N-terminus to its C-terminus, and the second polypeptide comprises a light chain of Fab1 and GD1 from its N-terminus to its C-terminus; or

[0093] (b) The first polypeptide comprises a heavy chain of Fab1 and GD1 from its N-terminus to its C-terminus, and the second polypeptide comprises a light chain of Fab1 and αCD3-VH1 or αCD3-VL1 from its N-terminus to its C-terminus.

[0094] In the embodiments disclosed herein, HB1 further includes a first Fc region. In these embodiments, the first Fc region is composed of a first Fc region subunit and a second Fc region subunit. In these embodiments, each Fc region subunit is composed of CH2 and CH3 domains.

[0095] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein the first Fc region is located between the C-terminus of the heavy chain of Fab1 and the N-terminus of αCD3-VH1 or αCD3-VL1. In alternative embodiments, the first Fc region is located at the C-terminus of αCD3-VH1 or αCD3-VL1.

[0096] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB1 is composed of a first polypeptide, a second polypeptide, and a third polypeptide, wherein

[0097] (a) The first polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0098] (i) The heavy chain of Fab1,

[0099] (ii) αCD3-VH1 or αCD3-VL1,

[0100] (iii) First Fc region subunit, and

[0101] (b) The second polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0102] (i) GD1

[0103] (ii) Second Fc region subunit, and

[0104] (c) The third polypeptide contains the light chain of Fab1.

[0105] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB1 is composed of a first polypeptide, a second polypeptide, and a third polypeptide, wherein

[0106] (a) The first polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0107] (i) The heavy chain of Fab1,

[0108] (ii) GD1,

[0109] (iii) First Fc region subunit, and

[0110] (b) The second polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0111] (i) αCD3-VH1 or αCD3-VL1

[0112] (ii) Second Fc region subunit, and

[0113] (c) The third polypeptide contains the light chain of Fab1.

[0114] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB1 is composed of a first polypeptide, a second polypeptide, and a third polypeptide, wherein

[0115] (a) The first polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0116] (i) The heavy chain of Fab1,

[0117] (ii) First Fc region subunit,

[0118] (iii) αCD3-VH1 or αCD3-VL1, and

[0119] (b) The second polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0120] (i) Second Fc region subunit,

[0121] (ii) GD1, and

[0122] (c) The third polypeptide contains the light chain of Fab1.

[0123] In the embodiments, this disclosure provides a pair of complementary hemimolecules, wherein HB1 further comprises a third Fab fragment (Fab3) that is specific to the first antigen (AG1).

[0124] In embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB1 is composed of a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein

[0125] (a) The first polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0126] (i) The heavy chain of Fab1,

[0127] (ii) αCD3-VH1 or αCD3-VL1,

[0128] (iii) First Fc region subunit, and

[0129] (b) The second polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0130] (i) Heavy chains of Fab3,

[0131] (ii) GD1,

[0132] (iii) Second Fc region subunit, and

[0133] (c) The third polypeptide contains the light chain of Fab1, and

[0134] (d) The fourth polypeptide contains the light chain of Fab3.

[0135] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB1 is composed of four polypeptides, wherein

[0136] (a) The first polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0137] (i) The heavy chain of Fab1,

[0138] (ii) First Fc region subunit,

[0139] (iii) αCD3-VH1 or αCD3-VL1, and

[0140] (b) The second polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0141] (i) Heavy chains of Fab3,

[0142] (ii) Second Fc region subunit,

[0143] (iii) GD1, and

[0144] (c) The third polypeptide contains the light chain of Fab1, and

[0145] (d) The fourth polypeptide contains the light chain of Fab3.

[0146] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein GD1 and αCD3-VH1 or GD1 and αCD3-VL1 are positioned adjacent to and parallel to each other on the first polypeptide and the second polypeptide.

[0147] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB2 is composed of a fifth polypeptide and a sixth polypeptide, wherein

[0148] (a) The fifth polypeptide comprises a heavy chain of Fab2 and αCD3-VH2 or αCD3-VL2 from its N-terminus to its C-terminus, and the sixth polypeptide comprises a light chain of Fab2 and optionally GD2 from its N-terminus to its C-terminus.

[0149] (b) The fifth polypeptide comprises a heavy chain of Fab2 and optionally GD2 from its N-terminus to its C-terminus, and the sixth polypeptide comprises a light chain of Fab2 and αCD3-VH2 or αCD3-VL2 from its N-terminus to its C-terminus.

[0150] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB2 further comprises a second Fc region consisting of a third Fc region subunit and a fourth Fc region subunit, wherein each Fc region subunit consists of CH2 and CH3 domains.

[0151] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein the second Fc region is located between the C-terminus of the heavy chain of Fab2 and the N-terminus of αCD3-VH2 or αCD3-VL2, respectively; or located at the C-terminus of αCD3-VH2 or αCD3-VL2.

[0152] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB2 is composed of a fifth polypeptide, a sixth polypeptide, and a seventh polypeptide, wherein

[0153] (a) The fifth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0154] (i) The heavy chain of Fab2,

[0155] (ii) αCD3-VH2 or αCD3-VL2,

[0156] (iii) The third Fc subunit, and

[0157] (b) The sixth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0158] (i) Optional GD2,

[0159] (ii) The fourth Fc subunit, and

[0160] (c) The seventh polypeptide contains the light chain of Fab2.

[0161] In the embodiments, this disclosure provides a pair of complementary half-molecules, wherein HB2 is composed of a fifth polypeptide, a sixth polypeptide, and a seventh polypeptide, wherein

[0162] (a) The fifth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0163] (i) The heavy chain of Fab2,

[0164] (ii) GD2,

[0165] (iii) The third Fc subunit, and

[0166] (b) The sixth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0167] (i) αCD3-VH2 or αCD3-VL2,

[0168] (ii) The fourth Fc subunit, and

[0169] (c) The seventh polypeptide contains the light chain of Fab2.

[0170] In the embodiments, this disclosure provides a pair of complementary hemimolecules, wherein HB2 is composed of three polypeptides, wherein

[0171] (a) The fifth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0172] (i) The heavy chain of Fab2,

[0173] (ii) Third Fc region subunit,

[0174] (iii) αCD3-VH2 or αCD3-VL2,

[0175] (b) The sixth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0176] (i) The fourth Fc subunit,

[0177] (ii) Choose GD2, and

[0178] (c) The seventh polypeptide contains the light chain of Fab2.

[0179] In the embodiments, this disclosure provides a pair of complementary hemimolecules, wherein HB2 further comprises a fourth Fab fragment (Fab4) that is specific to the second antigen (AG2).

[0180] In the embodiments, this disclosure provides a pair of complementary hemimolecules, wherein HB2 is composed of four polypeptides, wherein

[0181] (a) The fifth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0182] (i) The heavy chain of Fab2,

[0183] (ii) αCD3-VH2 or αCD3-VL2,

[0184] (iii) The third Fc subunit, and

[0185] (b) The sixth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0186] (i) The heavy chain of Fab4,

[0187] (ii) The fourth Fc subunit, and

[0188] (c) The seventh polypeptide contains the light chain of Fab2, and

[0189] (d) The eighth polypeptide contains the light chain of Fab4.

[0190] In the embodiments, this disclosure provides a pair of complementary hemimolecules, wherein HB2 is composed of four polypeptides, wherein

[0191] (a) The fifth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0192] (i) The heavy chain of Fab2,

[0193] (ii) αCD3-VH2 or αCD3-VL2,

[0194] (iii) The third Fc subunit, and

[0195] (b) The sixth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0196] (i) The heavy chain of Fab4,

[0197] (ii) αCD3-VH2 or αCD3-VL2, and

[0198] (iii) Fourth Fc subunit,

[0199] (c) The seventh polypeptide contains the light chain of Fab2, and

[0200] (d) This eighth polypeptide contains the light chain of Fab4.

[0201] If the fifth polypeptide contains αCD3-VH2, then the sixth polypeptide contains αCD3-VH2, or if the fifth polypeptide contains αCD3-VL2, then the sixth polypeptide contains αCD3-VL2.

[0202] In the embodiments, this disclosure provides a pair of complementary hemimolecules, wherein HB2 is composed of four polypeptides, wherein

[0203] (a) The fifth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0204] (i) The heavy chain of Fab2,

[0205] (ii) Third Fc region subunit,

[0206] (iii) αCD3-VH2 or αCD3-VL2, and

[0207] (b) The sixth polypeptide comprises, from its N-terminus to its C-terminus, the following...

[0208] (i) The heavy chain of Fab4,

[0209] (ii) Fourth Fc subunit,

[0210] (iii) Optionally αCD3-VH2 or αCD3-VL2; or optional GD2, and

[0211] (c) The seventh polypeptide contains the light chain of Fab2, and

[0212] (d) This eighth polypeptide contains the light chain of Fab4.

[0213] If the fifth polypeptide contains αCD3-VH2, then the sixth polypeptide contains αCD3-VH2 or GD2, or if the fifth polypeptide contains αCD3-VL2, then the sixth polypeptide contains αCD3-VL2 or GD2.

[0214] In the embodiments disclosed herein, the first Fc region and / or the second Fc region includes one or more amino acid modifications that promote association between the first Fc region subunit and the second Fc region subunit and / or the third Fc region subunit and the fourth Fc region subunit.

[0215] In the embodiments disclosed herein, in the CH3 domain of the first Fc subunit and / or the third Fc subunit, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and the serine residue at position 354 is replaced by a cysteine ​​residue (S354C); and in the CH3 domain of the second Fc subunit and / or the fourth Fc subunit, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), the threonine residue at position 366 is replaced by a serine residue (T366S), the leucine residue at position 368 is replaced by an alanine residue (L368A), and the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C); or vice versa, wherein the numbering is based on the EU index.

[0216] In the embodiments disclosed herein, the first Fc subunit, the second Fc subunit, the third Fc subunit, and the fourth Fc subunit contain one or more amino acid mutations that reduce the binding affinity of the Fc region to the Fc receptor and / or C1q, and / or reduce its effector function, wherein the one or more amino acid mutations are selected from the group consisting of: L234A, L235E, G237A, A330S, and P331S (EU number).

[0217] In the embodiments disclosed herein, Fab1, αCD3-VH1, αCD3-VL1, GD1, the first Fc region, and Fab3 on HB1 are connected to each other via peptide linkers. In the embodiments, Fab2, αCD3-VH2, αCD3-VL2, GD2, the second Fc region, and Fab4 on HB2 are connected to each other via peptide linkers.

[0218] In the embodiments disclosed herein, the peptide linker is an unstructured and / or flexible peptide linker. In some embodiments, the peptide linker is an incleavable peptide linker. In some embodiments, the peptide linker does not contain protease cleavage sites, particularly protease cleavage sites that can be cleaved by cancer-associated proteases. In some embodiments, the peptide linker contains an immunoglobulin hinge-derived sequence. In some embodiments, the peptide linker is composed of amino acid residues selected from the group consisting of: A, Q, D, P, H, G, S, E, T, K, and C. In the embodiments disclosed herein, the peptide linker is 1-50 amino acid residues in length.

[0219] In embodiments, this disclosure provides a pair of complementary hemimolecules for use as a medicine. In embodiments, this disclosure provides a pair of complementary hemimolecules for use in treating diseases associated with the presence of undesirable AG1 and AG2.

[0220] In embodiments, this disclosure provides a pharmaceutical composition comprising HB1 and a pharmaceutically acceptable carrier. In embodiments, this disclosure provides a pharmaceutical composition comprising HB2 and a pharmaceutically acceptable carrier. In embodiments, this disclosure provides a kit comprising a pharmaceutical composition containing HB1 and a pharmaceutical composition containing HB2. Attached Figure Description

[0221] Figure 1 Complementary CyCAT half-molecules in the form of B027, wherein the Fab fragment is fused to the N-terminus of a single αCD3-variable domain (αCD3-SVD, αCD3-VH, or αCD-VL) at the C-terminus of its heavy chain.

[0222] Figure 2 : Figure 2 A - Figure 2 F': Schematic illustration of various CyCAT half-forms lacking protected structural domains (GD) according to this disclosure. Each half-form is shown carrying either an αCD3-VH structural domain or an αCD3-VH structural domain.

[0223] Figure 2 A: CyCAT half-molecule in form B036, containing a Fab fragment, a single αCD3-variable domain (αCD3-SVD), and an Fc region containing a mortar-and-groove (KiH) mutation. The half-molecule has a backbone in which the single αCD3-SVD domain ( Figure 2 A') or a single αCD3-VL domain ( Figure 2 A) It fuses with the heavy chain of Fab at its N-terminus and with the Fc subunit carrying the club-shaped mutation at its C-terminus.

[0224] Figure 2 B: A CyCAT hemimolecule of form B063, comprising two identical Fab fragments, a single αCD3-SVD, and an Fc region containing a KiH mutation. The hemimolecule has a backbone in which either the αCD3-VH (B') or αCD3-VL domain (B) is fused at its N-terminus to the heavy chain of one Fab fragment and at its C-terminus to an Fc region subunit carrying a mortar-like mutation. A second Fc region subunit carrying a mortar-like mutation is coupled at its N-terminus to the heavy chain of a second Fab fragment within its CH3 domain.

[0225] Figure 2 The CyCAT half-molecule in form C:B039 comprises two identical Fab fragments, two identical αCD3-SVDs, and an Fc region. The half-molecule has two identical backbones, one of which has an αCD3-VH domain (C') or an αCD3-VL domain (C) fused at its N-terminus to the heavy chain of one Fab fragment and at its C-terminus to an Fc region subunit. Additionally, a second identical αCD3-VH domain (C') or a second identical αCD3-VL domain (C) is fused at its N-terminus to the heavy chain of a second Fab fragment and at its C-terminus to a second Fc region subunit. Due to its symmetrical structure, this form lacks the KiH mutation in its Fc region.

[0226] Figure 2 The CyCAT half-molecule in the form of D:B038 comprises a Fab fragment, an Fc region containing a KiH mutation, and a single αCD3-SVD. This half-molecule has a backbone in which the Fc region subunit carrying the KiH mutation is fused at its N-terminus to the heavy chain of the Fab fragment, and at its C-terminus to either a single αCD3-VH domain (D') or a single αCD3-VL domain (D).

[0227] Figure 2 The CyCAT half-molecule, in the form of E:B064, comprises two identical Fab segments, an Fc region containing a KiH mutation, and a single αCD3-SVD. The half-molecule has a main chain in which the Fc subunit carrying the KiH mutation is coupled at its N-terminus to the heavy chain of a Fab segment and fused at its C-terminus to either a single αCD3-VH domain (E') or a single αCD3-VL domain (E). The Fc subunit carrying the acetamipridoid mutation is fused at its N-terminus to the heavy chain of a second Fab segment.

[0228] Figure 2The CyCAT half-molecule in form F:B050 comprises two identical Fab fragments, an Fc region, and two identical αCD3-SVDs. The half-molecule has two identical backbones, one of which has an Fc region subunit fused at its N-terminus to the heavy chain of a Fab fragment and at its C-terminus to either an αCD3-VH domain (F') or an αCD3-VL domain (F). The other Fc region subunit is coupled at its N-terminus to the heavy chain of a second Fab fragment and at its C-terminus to either a second identical αCD3-VH domain (F') or a second identical αCD3-VL domain (F). Due to its symmetrical structure, this form lacks a KiH mutation in its Fc region.

[0229] Figure 3 Schematic illustrations of various CyCAT hemimorphic forms including a protective domain (GD) according to the present invention. In an example of the invention, the inactive antibody variable domain (GD) is... VH GD VH-杵 Or GD VL ) or HSA (GD HSAwt ) or HSA domain III (GD HSA-DIII The protective domain is used as a protective domain. When the protective domain is selected as an inactive antibody variable domain, it is chosen to have the same variable chain type as αCD3-SVD, i.e., in the case of the αCD3-VL domain, GD is used. VL Furthermore, in the case of the αCD3-VH domain, GD is used. VH .

[0230] Figure 3 A: A CyCAT half-molecule of form B073, comprising a Fab fragment, αCD3-SVD, a single protecting domain, and an Fc region containing a mortis mutation. The half-molecule has a backbone in which either the αCD3-VH domain (A') or the αCD3-VL domain (A) is coupled to the heavy chain of the Fab fragment via its N-terminus and to the Fc region subunit carrying the mortis mutation via its C-terminus. The protecting domain is coupled to a second Fc region subunit carrying the mortis mutation via its C-terminus, such that the corresponding αCD3-SVD and GD are arranged parallel to each other and adjacent to each other.

[0231] Figure 3B: CyCAT hemimolecule in form B099, an alternative embodiment to form B073. The αCD3-SVD domain and a single GD are interchanged on the two polypeptide chains. The hemimolecule has a backbone in which the GD is coupled to the heavy chain of the Fab fragment via its N-terminus and to the Fc subunit carrying the mortis mutation via its C-terminus. The αCD3-VH domain (B') or the αCD3-VL domain (B) is further coupled to the second Fc subunit carrying the mortis mutation via its C-terminus.

[0232] Figure 3 The CyCAT hemimolecule in the form of C:B077 comprises two Fab fragments, a single αCD3-SVD, a single protecting domain, and an Fc region. The hemimolecule has two distinct backbones, one of which, an αCD3-VH domain (C') or an αCD3-VL domain (C), is coupled via its N-terminus to the heavy chain of one Fab fragment and via its C-terminus to an Fc subunit carrying a galvanic mutation. The single GD is coupled via its N-terminus to the heavy chain of the second Fab fragment and via its C-terminus to a second Fc subunit carrying a galvanic mutation, such that the corresponding αCD3-SVD and GD are arranged parallel to each other and adjacent to one another.

[0233] Figure 3 The CyCAT half-molecule in the form of D:B103 comprises a Fab fragment, an Fc region, a single αCD3-SVD, and a single protecting domain (GD). The half-molecule has a main chain in which the Fc region subunit carrying the mortar-mutant is coupled at its N-terminus to the heavy chain of the Fab fragment and at its C-terminus to either a single αCD3-VH domain (D') or a single αCD3-VL domain (D). The GD is coupled via its N-terminus to the C-terminus of the Fc region subunit carrying the mortar-mutant, such that the single αCD3-SVD and GD are arranged parallel to each other and adjacent to one another.

[0234] Figure 3 The CyCAT half-molecule, in the form of E:B101, comprises two Fab segments, an Fc region, a single αCD3-SVD, and a single protecting domain. The half-molecule has two main chains. In one main chain, the Fc subunit carrying the galvanic mutation is coupled to the heavy chain of one Fab segment via its N-terminus and to either a single αCD3-VH domain (E') or a single αCD3-VL domain (E) via its C-terminus. In the second main chain, the Fc subunit carrying the galvanic mutation is coupled to the heavy chain of the second Fab segment via its N-terminus and to the protecting domain via its C-terminus, such that the αCD3-SVD and GD are arranged parallel to each other and adjacent to one another.

[0235] Figure 4Illustration of the formation of a complex of two complementary CyCAT hemimolecules, one of which contains a protective domain. Co-localization of the two complementary hemimolecules on the target cell leads to the displacement of GD and the formation of a functional Fv-binding fragment capable of binding to CD3 on the surface of T cells.

[0236] Figure 5 Exemplary results of cytotoxicity assays for determining the assay window of dual-targeting half-pairs are shown. Results are presented according to dual-targeting half-pairs: 1, single-targeting half-pairs: 2, dual-targeting half-pairs: 3, and single-targeting half-pairs: 4 in Table 8. Detailed Implementation

[0237] definition

[0238] As used herein, the terms “first,” “second,” “third,” “fourth,” “fifth,” “sixth,” “seventh,” “eighth,” etc., relating to target antigens, halves, antibodies, antibody fragments, Fab, Fv region, Fc region, Fc subunit, peptide linkers, or polypeptides, are used to distinguish cases where more than one of each type of component exists. Unless explicitly stated otherwise, the use of these terms is not intended to assign a particular order or direction.

[0239] "Administered" includes, but is not limited to, delivery of the drug via injectable forms (e.g., intravenous, intradermal, or subcutaneous routes) or mucosal routes (e.g., as a nasal spray or aerosol for inhalation) or as an ingestible solution, capsule, or tablet. Preferably, administration is via injectable forms.

[0240] As used herein, the term "affinity" refers to the strength of the interaction between a peptide and its target at a single site. Within each site, the peptide's binding region interacts with its target at many sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0241] As used herein, the term "antigen" or "target antigen" refers to any target molecule that can bind to one of the binding sites present in an antibody. Typically, an antigen is a peptide, protein, or any other protein molecule. Alternatively, an antigen can be any other organic or inorganic molecule, such as a carbohydrate, fatty acid, lipid, dye, or fluorophore.

[0242] As used herein, “amino acid residue” or “amino acid” will be indicated by their full name or according to standard three-letter or one-letter amino acid codes. “Naturally occurring amino acids” refers to the following amino acids:

[0243] Table 1: Naturally occurring amino acids

[0244]

[0245] As used herein, the term "antibody" or "immunoglobulin" (Ig) molecule refers to a protein containing at least two heavy (H) chains and two light (L) chains that interacts with an antigen. Each heavy chain (HC) consists of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain (LC) consists of a light chain variable domain (abbreviated as VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL domains 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 from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable domains (VH and VL) of the heavy and light chains contain or form "binding sites" or "antigen-binding sites" that selectively interact with or bind to antigens. These constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies can belong to any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into regions with structural and functional homology.The structure and location of immunoglobulin variable domains (e.g., CDRs) can be defined using well-known numbering schemes (e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia numbering schemes) (see, for example, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), edited by Kabat et al.; Lazikani et al., (1997) J. Mol. Biol. [Journal of Molecular Biology] 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242 USD; 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 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948. As used herein, the term “antibody” is intended to include antibody fragments, CyCAT molecules, hemimolecules, monospecific antibodies, bispecific antibodies, and multispecific antibodies.

[0246] As used herein, the term “antibody fragment” refers to one or more portions of an antibody that retain the ability to interact specifically with an antigen (e.g., through binding, steric hindrance, or stable spatial distribution). Examples of antibody fragments include, but are not limited to, Fab fragments (“Fab”), monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments, which are bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments (“Fv”) consisting of the VL and VH domains of a single arm of the antibody; dAb fragments consisting of the VH domain (Ward et al., 1989, Nature 341:544-546); and separated complementarity-determining regions (CDRs). Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, they can be produced using recombination methods via a synthetic linker that allows them to be produced as a single protein chain, where the VL and VH domains pair to form a monovalent molecule (referred to as a single-chain Fv (“scFv”); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be covered within the scope of the term “antibody fragment.” Antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for efficacy in the same manner as intact antibodies. Antibody fragments can also be incorporated into single-domain antibodies, macrobodies, minibodies, intracellular antibodies, biantibodies, triantibodies, tetraantibodies, v-NARs, and biscFvs (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 a fibronectin peptide monomer). Antibody fragments can be incorporated into single-chain molecules containing a pair of tandem Fv fragments to form a pair of antigen-binding sites with complementary light chain peptides (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Patent No. 5,641,870).

[0247] As used herein, the terms "binding site," "antigen binding site," or "antigen binding region" refer to a protein-formed structure capable of binding to or specifically binding to an antigen. A binding site need not be a series of consecutive amino acids, or even an amino acid within a single polypeptide chain. For example, in an Fv generated from two different polypeptide chains, the "binding site" consists of a series of amino acids, VL and VH, that interact with the antigen and are typically (but not always) located within one or more CDRs in each variable region. In some embodiments, the "binding site" is or comprises, or is formed by, a complementary pair of antibody variable heavy chains (VH) and light chains (VL). The VH and V forming the binding site can be in a single polypeptide chain or in different polypeptide chains. In preferred embodiments, the binding site is or comprises, is formed by, or is formed by, a VH present on a first hemimolecule according to this disclosure and a complementary VL present on a second hemimolecule according to this disclosure, or vice versa. In some embodiments, the "binding site" has one VH and one VL. In some embodiments, the binding site comprises one or more CDRs of the antibody. In other embodiments, the binding site is derived from antibody mimics, such as affinity molecules, alphabets, antiicalins, avimers, DARPins, fynomers, Kunitz domain peptides, helical-turn-helical peptides, or monoclonal antibodies.

[0248] As used herein, the terms “specifically binds to,” “specifically binds to,” “specifically identifies,” or “specifically recognizes” refer to measurable and reproducible interactions, such as the binding between a target antigen and an antibody, antibody fragment, or hemimolecule disclosed herein, which determine the presence of the target antigen in the presence of a heterogeneous group of molecules, including biomolecules. For example, an antibody, antibody fragment, or hemimolecule disclosed herein that specifically binds to a target antigen (which may be an antigen or an epitope of an antigen) binds to that target with a higher affinity, stronger affinity, easier binding, and / or longer duration than it binds to other target antigens. In some embodiments, the antibody, antibody fragment, or hemimolecule specifically binds to an epitope on a conserved protein from proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding. The antibodies, antibody fragments, or hemimolecules disclosed herein specifically bind to antigens. Methods for determining whether two molecules specifically bind are well known in the art, such as standard ELISA assays. Scoring can be performed using standard colorimetric methods (e.g., using horseradish peroxide for secondary antibodies and hydrogen peroxide for tetramethylbenzidine). The reaction in certain wells is scored using optical density (e.g., at 450 nm). A typical background (=negative reaction) can be 0.1 OD; a typical positive reaction can be 1 OD. This means the difference between positive and negative can exceed 5-fold. Typically, binding specificity is determined not by using a single reference antigen, but by using a set of three to five unrelated antigens (e.g., infant formula, BSA, transferrin, etc.).

[0249] "CD3" refers to an antigen that is expressed on T cells as part of a multimolecular T cell receptor (TCR) and consists of homodimers or heterodimers formed by the association of two of the following four receptor chains: CD3ε (CD3e), CD3δ, CD3ζ, and CD3γ.

[0250] The amino acid sequence of human CD3ε (or human CD3e) is UniProt P07766:

[0251] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:19) (signal sequence underlined, intracellular regions in italics, transmembrane regions in bold).

[0252] The mature extracellular domain of human CD3ε (excluding the signal sequence) contains the following amino acid sequence:

[0253] QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 20)

[0254] The amino acid sequence of cynomolgus monkey CD3ε (or cynomolgus monkey CD3e or cynomolgus monkey CD3e) is UniProt Q95LI5:

[0255] MQSGTRWRVLGLCLLSIGVWG QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI (SEQ ID NO: 21) (signal sequence underlined, intracellular region italicized, transmembrane region bold).

[0256] The mature extracellular region (excluding the signal sequence) of CD3ε in cynomolgus monkeys contains the following amino acid sequence:

[0257] QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI (SEQ ID NO: 22).

[0258] The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody having a constant antibody region derived from or corresponding to a sequence found in one species and a variable antibody region derived from another species. Preferably, the constant antibody region is derived from or corresponds to a sequence found in humans, and the variable antibody region (e.g., VH, VL, CDR, or FR regions) is derived from a sequence found in non-human animals (e.g., mice, rats, rabbits, or hamsters).

[0259] As used herein, the terms “CyCAT hemimolecule,” “hemimolecule,” or “HB” or “hemi” in their broadest sense refer to an antigen-binding molecule that specifically binds to at least one target antigen and consists of an antibody fragment (e.g., a Fab fragment) and a VH or VL of an antibody Fv fragment specific to CD3. Therefore, the hemimolecule according to this disclosure incorporates a CD3-specific half-antibody Fv-binding fragment. The essential characteristics of the hemimolecule according to this disclosure are described in WO 2013 / 104804 (University of Würzburg, Julius-Maximilians-Universität Würzburg) (which is incorporated herein in its entirety).

[0260] As used herein, the term “CyCAT molecule” refers to a functional complex of two complementary CyCAT hemimolecules as described herein, wherein each hemimolecule carries half of an antibody Fv fragment that is specific to CD3, and wherein only the complex can function the Fv fragment, while a single CyCAT hemimolecule does not possess this function on its own.

[0261] As used herein, a “complementary” CyCAT hemimolecule means any complex or pair of two CyCAT hemimolecules as described herein; provided that one CyCAT hemimolecule carries at least one single αCD3-VH or αCD3-VL domain of the αCD3-Fv fragment, and the second CyCAT hemimolecule accordingly carries at least one complementary unpaired αCD3-VL or αCD3-VH domain of that αCD3-Fv fragment. When two complementary CyCAT hemimolecules bind to their target antigen present on the surface of the same target cell, the αCD3-VH and αCD3-VL domains dimerize and become functionally complementary.

[0262] As used in this article, a "covalent bond" refers to an interatomic bond characterized by the sharing of electrons.

[0263] As used in this article, the term "EC" 50 "CyCAT" refers to the concentration of the antibody, antibody fragment, or CyCAT molecule that induces a half-maximal response between baseline and maximum value in the assay. Therefore, it represents the concentration at which 50% of the maximum effect is observed.

[0264] As used herein, “human antibody” or “human antibody fragment” includes antibodies and antibody fragments having variable regions, both of which, including the frame region and CDR region, are derived from human-derived sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from such a sequence. Human-derived sequences include, for example, human germline sequences, or mutant forms of human germline sequences, or antibodies containing a common frame sequence derived from human frame sequence analysis, for example, as described by 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., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia numbering schemes) (see, for example, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), edited by Kabat et al.; Lazikani et al., (1997) J. Mol. Biol. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edition, NIH Publication No. 91-3242 US Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature [Nature] 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. [Journal of Molecular Biology] 273:927-948. Human antibodies and human variable regions can also be isolated from synthetic libraries or from transgenic mice (e.g., xenomouse), provided that the antibodies produced by each system have variable regions, and both the framework region and CDR region of these variable regions are derived from human-derived sequences.

[0265] "Humanized antibody" or "humanized antibody fragment" is defined herein as an antibody having a constant antibody region and a variable antibody region or a portion thereof derived from a human-derived sequence, or having only a CDR derived from another species. Humanization can be achieved through a variety of methods, including but not limited to (a) transplanting a non-human (e.g., donor antibody) CDR onto a human (e.g., receptor antibody) framework and constant region with or without retaining key framework residues (e.g., those residues important for maintaining good antigen-binding affinity or antibody function); (b) transplanting only non-human specificity-determining regions (SDRs or a-CDRs; residues essential for antibody-antigen interactions) onto a human framework and constant region; or (c) transplanting the entire non-human variable domain, but "covering" them with human-like segments by replacing surface residues.Humanized antibodies and their preparation methods are reviewed in, for example, Almagro and Fransson, Front Biosci, 13, 1619-1633 (2008), and further described in, for example, Riechmann et al., Nature, 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA, 86, 10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Jones et al., Nature, 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci, 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, MolecImmun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) transplantation); Padlan, Mol Immunol 28, 489-498 (1991) (describing “surface reconstruction”); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing “FR reorganization”); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer [British Journal of Cancer] 83, 252-260 (2000) (describing the “guided selection” approach to FR reorganization).

[0266] As used in this article, the term "IC" 50 "CyCAT" refers to the concentration of an antibody, antibody fragment, or CyCAT molecule that inhibits half of the response between the maximum response and baseline in an assay. Therefore, it represents the concentration that reduces a given response by 50%.

[0267] The term "isolated" refers to a compound (which may be, for example, an antibody, antibody fragment, or hemimolecule) that is substantially free of other antibodies, antibody fragments, or hemimolecules with different antigen specificities. Furthermore, isolated antibodies, antibody fragments, or hemimolecules may be substantially free of other cellular material and / or chemicals. Therefore, in some embodiments, the antibodies, antibody fragments, or hemimolecules provided herein are isolated antibodies, antibody fragments, or hemimolecules that have been separated from antibodies or hemimolecules with different specificities. Isolated antibodies or hemimolecules may be monoclonal antibodies, antibody fragments, or hemimolecules. Isolated antibodies, antibody fragments, or hemimolecules may be recombinant monoclonal antibodies, antibody fragments, or hemimolecules. However, isolated antibodies, antibody fragments, or hemimolecules that specifically bind to a target epitope, isotype, or variant may be cross-reactive with other relevant antigens (e.g., from other species, such as species homologs).

[0268] The terms “inhibition” or “inhibit,” “reduction” or “neutralization” or “neutralize” refer to a reduction or cessation of any phenotypic characteristic (e.g., binding, biological activity, or function), or a reduction or cessation of the occurrence, extent, or likelihood of that characteristic. “Inhibition” need not be complete, provided it can be detected using appropriate assays. In some embodiments, “reduction” or “inhibition” means the ability to result in a reduction of 20% or greater. In another embodiment, “reduction” or “inhibition” means the ability to result in a reduction of 50% or greater. In yet another embodiment, “reduction” or “inhibition” means the ability to result in an overall reduction of 75%, 85%, 90%, 95%, or greater.

[0269] As used in this article, the term "K" D "" refers to the dissociation constant, which is obtained from K d With K a The ratio (i.e., K) d / K a And expressed as molar concentration (M). The KB of an antigen-binding moiety (like, for example, a monoclonal antibody) can be determined using methods well-established in the art. D Value. The K value of the antigen-binding moiety (such as a monoclonal antibody) is measured. D The methods include SET (Soluble Equilibrium Titration), surface plasmon resonance using a biosensor system (such as the Biacore® system), or biolayer interferometry (BLI).

[0270] As used herein, the terms “monoclonal antibody,” “monoclonal antibody fragment,” or “monoclonal hemimolecule” refer to the antibody, antibody fragment, or hemimolecule disclosed herein derived from a single clone, including any eukaryotic clone, prokaryotic clone, or phage clone, and not to the method of its production. Monoclonal antibodies or antibody fragments can be prepared by hybridoma methods as described in Kohler et al.; Nature, 256:495 (1975) or can be isolated from a phage library. Other methods for preparing the clonal cell lines disclosed herein and the monoclonal antibodies or hemimolecules expressed therefrom are well known in the art (see, for example, Chapter 11, in: Short Protocols in Molecular Biology, (2002) 5th Edition, Ausubel et al., editors, John Wiley and Sons, New York).

[0271] The term "multispecific" means that an antibody, half-molecule, or CyCAT molecule can specifically bind to two or more different antigens. Typically, a multispecific antibody, half-molecule, or CyCAT molecule consists of two or more antigen-binding sites, each specific to a different antigen or epitope. The term "bispecific" means that an antibody, half-molecule, or CyCAT molecule can specifically bind to two different antigens. Typically, a bispecific antibody, half-molecule, or CyCAT molecule contains two antigen-binding sites, each specific to a different antigen or epitope. The term "trispecific" means that an antibody, half-molecule, or CyCAT molecule can specifically bind to three different antigens. Typically, a trispecific antibody, half-molecule, or CyCAT molecule contains three antigen-binding sites, each specific to a different antigen or epitope.

[0272] The term "pharmaceuticalally acceptable carrier" refers to a component of a pharmaceutical composition that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0273] As used in this article, "non-covalent association" refers to molecular interactions that do not involve interatomic bonds. Non-covalent interactions include, for example, ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces.

[0274] The term "pharmaceutical composition" refers to a formulation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that the formulation does not contain any other components that would be unacceptably toxic to a subject administering the formulation.

[0275] As used herein, the term "peptide" refers to a polymer of amino acid residues and not to a product of a specific length. This term applies to both naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, the specific amino acid sequence of a peptide also implicitly encompasses variants of its conserved modifications (e.g., by replacing an amino acid residue with another amino acid residue having similar structure and / or chemical properties). Peptides can be derived from natural biological sources or produced through recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be generated in any manner, including through chemical synthesis. Peptides may also contain one or more disulfide bonds.

[0276] As used herein, the terms “recombinant antibody,” “recombinant antibody fragment,” or “recombinant hemimolecule” include all antibodies, antibody fragments, or hemimolecules prepared, expressed, generated, or isolated according to this disclosure by means not found in nature. For example, antibodies or hemimolecules isolated from host cells transformed to express antibodies or hemimolecules; antibodies selected and isolated from recombinant, combined human antibody libraries; and antibodies prepared, expressed, generated, or isolated by any other means (involving the splicing of all or part of the human immunoglobulin gene or sequence into other DNA sequences), or antibodies isolated from animals (e.g., mice) where the human immunoglobulin gene is transgenic or transchromosomally modified, or from hybridomas prepared from such animals. Preferably, such recombinant antibodies or hemimolecules have variable regions, in which the frame region and CDR region are derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies or hemimolecules may be mutagenized in vitro (or in vivo somatic mutagenesis when using animals that are transgenic for human Ig sequences), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody or hemimolecule are sequences that, although derived from and associated with human germline VH and VL sequences, may not naturally exist in an in vivo human antibody germline library. The recombinant antibody or hemimolecule may be a recombinant monoclonal antibody or a recombinant monoclonal hemimolecule. In the embodiments, the antibodies and antibody fragments disclosed herein are isolated from Ylanthia® antibody libraries disclosed in, such as US 13 / 321,564 or US 13 / 299,367 (both of which are incorporated herein by reference).

[0277] As used herein, “species” means any mammal, including rodents (e.g., mice or rats) and primates (e.g., cynomolgus monkeys, rhesus monkeys, or humans). Preferably, the subject is a primate, and most preferably a human.

[0278] The "therapeutic effective amount" or "effective amount" of a pharmaceutical agent (such as a pharmaceutical composition) refers to the amount that effectively achieves the desired therapeutic or preventive outcome at the necessary dosage and duration. A therapeutically effective amount of a pharmaceutical agent may eliminate, reduce, delay, minimize, or prevent adverse reactions to a disease.

[0279] As used herein, “treatment (such as treatment, treat, or treating)” means a clinical intervention that attempts to alter the natural course of a disease in an individual being treated and may be performed for prevention or during a clinicopathological process. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the hemimolecules according to this disclosure are used to delay the development of disease or slow its progression. Example

[0280] This invention relates to methods for reducing the toxicity and on-target or off-target side effects of bispecific or multispecific antibodies (including antibody-like functional proteins or biologics) that co-conjugate T cells to destroy tumor cells. These methods are performed by activating their CD3 binding capacity only near cancerous tissue, thereby avoiding off-target and on-target interactions. The combined method of this invention uses two complementary bifunctional hemimolecules, each targeting two different tumor-associated antigens. Each of the two CyCAT hemimolecules carries half of the αCD3-Fv binding fragment, and more specifically, carries either its VH or VL portion.

[0281] As used herein, complementary CyCAT hemimolecules refer to any pair of CyCAT hemimolecules as described herein; provided that one CyCAT hemimolecule carries at least one single αCD3-VH or αCD3-VL domain of the αCD3 Fv fragment, and the second CyCAT hemimolecule correspondingly carries at least one single complementary unpaired αCD3-VH or αCD3-VL domain of the αCD3 Fv fragment, which dimerize to form a functional αCD3-Fv binding fragment. Functional VH / VL CD3 specificity can only be generated through cellular complementarity when the two complementary CyCAT hemimolecules bind to the same target cell expressing both target antigens on its cell surface. This combination principle enhances the specificity of both the targeting and effector processes, thus addressing a core issue in T cell activation of bispecific antibodies.

[0282] Therefore, this invention relates to complementary pairs of hemimolecules (such hemimolecules are each referred to herein as "CyCAT hemimolecules" or "CyCAT hemimolecules" or "hemimolecules" or simply "HB"). Each hemimolecule is capable of binding to a target antigen expressed on the cell surface (e.g., a tumor-associated antigen expressed on cancer cells), but because they carry only half of a functional αCD3-Fv antibody fragment, they cannot recruit T cells to destroy tumor cells on their own. Each hemimolecule according to this disclosure has a plurality of functional protein components that can fuse with each other via peptide linkers. Another component is a single or two identical antibody variable domains of a CD3-specific antibody (e.g., a single VH domain or two identical VH domains or a single VL domain or two identical VL domains), which binds to CD3 to form a functional αCD3 Fv binding domain when CD3 is in close proximity to its complementary or homologous antibody variable domain. In this regard, an active CD3-binding domain (and therefore the trispecific antibody disclosed herein) forms on the surface of tumor cells expressing two targeting tumor antigens. Further components of such a hemimolecule may include an IgG Fc region that allows for an extended half-life. The IgG Fc region may further include an immunoglobulin hinge region at its C-terminus, which further stabilizes the heterodimeric Fc region by forming disulfide bonds.

[0283] Protected structural domains and halves containing such protected structural domains

[0284] To enhance the therapeutic safety profile of the CyCAT method described herein, this invention aims to minimize the amount of any complexes formed between the two complementary hemimolecules in the absence of target cells with both target antigens on their cell surface. Even small amounts of such complexes or aggregates can lead to both on-target and off-target T cell activation. Therefore, it is important to avoid any such unwanted heterodimerization by selecting appropriate hemimolecule structures. Such on-target and off-target induced heterodimerization can result from the inherent low-affinity binding (hydrophobic interaction) of the antibody VH and VL domains under conditions of localized high concentrations of the complementary CyCAT hemimolecules. For example, this may occur shortly after or during the simultaneous administration of a high concentration of the hemimolecule composition. To achieve such an increase in the "assay window" or "therapeutic window," this invention utilizes at least one protective domain (GD) covalently fused to at least one of the two complementary hemimolecules.

[0285] In the context of this disclosure, the “protective domain” is positioned on the hemisome in such a way that, in the absence of cells expressing both target antigens on their cell surface, the protective domain spatially blocks or shields the unpaired αCD3-VH or αCD3-VL domain, preventing it from binding to its complementary unpaired αCD3-SVD (αCD3-VH or αCD3-VL) present on the second hemisome molecule. However, this shielding effect of the protective domain as described herein is only effective in the absence of target cells expressing both target antigens; once the two complementary CyCAT hemisome molecules bind to their target antigens on the surface of such target cells, this shielding effect is lost. Based on the evidence provided in the examples herein, it is shown that once the two complementary hemisome molecules are close to each other on the surface of cells expressing both target antigens, GD displaces from the shielded αCD3-SVD, thereby allowing the formation of a functional αCD3-Fv domain (see also the present application). Figure 4 In this sense, GD does not function as a specific binding or mismatched partner of the αCD3-SVD to be shielded, and therefore does not require its complete release from the half-molecule, for example, through proteolytic cleavage.

[0286] The hemimolecule disclosed herein is designed such that complementary αCD3-VH and αCD3-VL domains exhibit minimal association / dimerization in solution over a wide concentration range in the absence of target cells, but exhibit unhindered association / dimerization in the presence of such target cells without requiring the release of the GD domain from the hemimolecule. As described above, this is achieved by means other than the binding of the protective domain to the unpaired αCD3-SVD (e.g., through their size and proximity).

[0287] Therefore, the protective domain according to this disclosure can be any protein or polypeptide of sufficient molecular size to provide steric blocking of αCD3-SVD when located at a site near such a target αCD3-SVD, without inhibiting or interfering with the specific binding of the target Fab fragment, nor allowing GD to bind to any other component of the hemimolecule to which it is attached. In particular, it neither binds to or associates with αCD3-SVD present on the same hemimolecule to be shielded, nor forms a functional Fv domain with any other component present on either of the two complementary hemimolecules.

[0288] In the embodiments disclosed herein, the GD does not bind to the VH (αCD3-VH) or VL (αCD3-VL) of the CyCAT hemimolecule as described herein. In the embodiments, the GD does not specifically bind to the αCD3-VH or αCD3-VL of the CD3-specific Fv present on the same CyCAT hemimolecule. In the embodiments disclosed herein, the GD does not bind to αCD3-SVD. In the embodiments, the GD does not specifically bind to αCD3-SVD. In the embodiments, the GD does not specifically bind to αCD3-VH or αCD3-VL. Non-binding in this disclosure does not exclude non-specific binding or low levels of residual binding (e.g., < 1%, < 5%, < 10%).

[0289] In embodiments disclosed herein, GD is present in, is a portion of, or is fused to one of the two complementary hemimolecules according to this disclosure. In embodiments, the GD is present in, is a portion of, or is fused to each of the two complementary hemimolecules according to this disclosure. In embodiments, GD does not bind to any component of the CyCAT hemimolecule. In embodiments, GD does not bind to any component of the hemimolecule fused to it. In embodiments, the component is selected from the group consisting of: targeting portions (e.g., Fab fragments), antibody variable regions, antibody constant domains, Fc regions, Fc region subunits, and peptide linkers.

[0290] The protecting domains disclosed herein are used in several different ways, depending on the structure of the CyCAT hemimolecule, as described herein and as... Figure 3 As shown.

[0291] In cases where the CyCAT hemimolecule is intended for use in humans, GD is preferably selected to be composed of human proteins or predominantly human proteins to minimize the risk of immunogenic reactions in humans.

[0292] In some embodiments, the GD domain according to this disclosure is a protein. In some embodiments, GD is a polypeptide. In some embodiments, GD is a human protein or human polypeptide, or a protein or polypeptide of human origin, or derived from a human protein or human polypeptide, or composed primarily of a human protein or human polypeptide.

[0293] In some embodiments, the molecular weight of the GD according to this disclosure is between 10 kDa and 110 kDa, for example, 20 kDa to 100 kDa, such as 25 kDa to 90 kDa, 30 kDa to 85 kDa, 35 kDa to 75 kDa, or 40 kDa to 70 kDa. In some embodiments, the molecular weight of the GD is less than 100 kDa, for example, 95 kDa, 90 kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa, 65 kDa, 60 kDa, 55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa, 25 kDa, 20 kDa, 15 kDa, or 10 kDa. In an embodiment, the molecular weight of the GD disclosed herein is less than 70 kDa. In an embodiment, the molecular weight of the GD disclosed herein is about 10 kDa to about 70 kDa.

[0294] Instances of protected structural domains

[0295] Examples of GDs suitable for the purposes of this invention that shield individual αCD3-VH or αCD3-VL domains to prevent them from binding to each other include globular proteins such as serum albumin, transferrin (Trf), and immunoglobulin domains.

[0296] In this embodiment, GD is a water-soluble globular protein or a spherical protein. In this embodiment, GD is selected from, but is not limited to, the group consisting of: serum albumin (SA), transferrin (Trf), portions or fragments of serum albumin or transferrin, and immunoglobulin domains. In this embodiment, the immunoglobulin domain is an antibody variable domain, such as a VH or VL domain, or an IgG constant domain, such as a CH1, CH2, CH3, or CH4 domain. In this embodiment, GD is an antibody variable domain. In this embodiment, GD is serum albumin, preferably human serum albumin (HSA) or a fragment thereof.

[0297] Antibody variable domains as protective domains

[0298] In the embodiments, according to this disclosure, GD is an antibody variable domain (GD). VD There are many GDs that can be used in this invention. VD .

[0299] GD or GD VD It may include a VH or VL with a frame region (preferably a human frame region or a human shared frame region) and an "inactive" CDR (which, when paired with a complementary antibody variable domain, does not form a functional antibody Fv domain capable of specifically binding to any target antigen). In the examples, GD is an inactive antibody variable domain (GD... VDIn the embodiments, GD is the inactive antibody variable heavy chain domain (GD). VH In the embodiments, GD is the inactive antibody variable light chain domain (GD). VL To prevent GD via αCD3-VH. VL Or αCD3-VL and GD VH Dimerization within the molecule forms an inactive Fv fragment (i.e., unable to specifically bind to the target antigen), and a half-molecule preferably utilizes the antibody variable domain of the same chain type. Therefore, possible combinations include: αCD3-VH and GD... VH Or αCD3-VL and GD VL .

[0300] In some embodiments, when the VH (αCD3-VH) or VL (αCD3-VL) domain of the CD3-specific Fv (αCD3-Fv) is αCD3-VH, then GD is GD. VH Furthermore, if αCD3-VH or αCD3-VL is VL, then GD is GD. VL In some embodiments, when αCD3-SVD is αCD3-VH, GD is GD. VH And when αCD3-SVD is αCD3-VL, GD is GD. VL GD was chosen as GD VD In this case, it can have the same sequence as or a different sequence from the αCD3-VH or αCD3-VL to be shielded. Preferably, GD VD The selected sequence has a different sequence from αCD3-SVD. In the embodiment, GD VD It has the same sequence as αCD3-SVD. In the embodiment, GD VD It has a sequence different from αCD3-SVD.

[0301] Therefore, GD VH and / or GD VL Each can independently contain one or more mutations that prevent specific binding to target antigens with such protective domains by (i) itself or (ii) when paired with a complementary inactive variable domain, or (iii) when paired with an active αCD3-SVD.

[0302] The Fv fragments formed as described in (i) to (iii) above may be referred to herein as “inactive Fv fragments” or “Fv fragments”. 无活性 That is, in (a) GD VH and GD VL (b) GD VHand αCD3-VL, or (c) αCD3-VH and GD VL The Fv segment formed between them.

[0303] Because the same type of antibody variable chain is used on the same hemimolecule for αCD3-SVD and GD combination (i.e., αCD3-VH and GD) VH or αCD3 VL With GD-VL), and in conjunction with GD VD The inactive CDR is used, therefore the half-molecule itself cannot bind to CD3 according to this disclosure.

[0304] Additionally, GD exists on a half-molecule VH The structural domain and GD present on the complementary half-molecule VL After association, they can only form inactive Fv domains that cannot bind to the target antigen. According to the preferred GD disclosed herein... VH and GD VL This is achieved through mutations in the wild-type or parental VH or VL domain sequences, as further outlined below. Exemplary mutations are located in the CDR1, CDR2, or CDR3 regions of the corresponding VH or VL domain.

[0305] In a preferred embodiment, GD VD The sequence of the frame region of GD is identical or identical to the sequence of the frame region of the αCD3-SVD to be masked. That is, for example, if the frame region of αCD3-VH contains the germline protein sequence of VH1-18, then GD... VHThe framework region also contains the germline protein sequences of VH1-18. The “germ protein sequence” or “germ amino acid sequence” of antibodies encoded by germline genes is disclosed, for example, in the following publications: For VH: Tomlinson et al., (1992), “The Repertoire of Human Germline Vh Sequences Reveals about Fifty Groups of VhSegments with Different Hypervariable Loop” J. Mol. Biol. 227, 776-798; Matsuda et al. (1998); “The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus” JExp Med 188(11):2151-62; and LeFranc MP (2001) “Nomenclature of the human immunoglobulin heavy (IGH) genes”. [Nomenclature of the human immunoglobulin heavy chain (IGH) gene].”Exp Clin Immunogenet. [Experimental and Clinical Immunogenetics] 18(2):100-16; For Vλ: Kawasaki et al., (1997) “One-Megabase Sequence Analysis of the Human immunoglobulin lambda Gene Locus” Genome Research 7(3):250-61; Frippiat et al., (1995) “Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2” Hum. Mol. Genet.[Human Molecular Genetics], 4, 983-991; and LeFranc MP (2001) “Nomenclature of the human immunoglobulin lambda (IGL) genes.”Exp ClinImmunogenet. [Experimental and Clinical Immunogenetics]; 18:242-254; and for Vκ: Schäble and Zachau (1993), “The variable genes of the human immunoglobulin kappa locus,” Biol. Chem Hoppe Seyler. [Hoppe Seyler Biochemistry] 374(11):1001-22; Brensing-Küppers et al. (1997), “The human immunoglobulin kappa locus on yeast artificial chromosomes (YACs)” Gene. [Genes] 191(2):173-81; Kawasaki et al. (2001), “Evolutionary dynamics of the human immunoglobulin kappa locus and the germline repertoire of the Vkappa genes” Eur J Immunol 31(4):1017-28; and Lefranc MP (2001) “Nomenclature of the human immunoglobulin kappa (IGK) genes” ExpClin Immunogenet. 18, 161-174, all of which are incorporated herein by reference in their entirety.

[0306] In some embodiments, inactive GD according to this disclosure VDThe generation of inactivation is typically accomplished by altering one or more CDRs in the VH and / or VL of an antibody (e.g., an αCD3 antibody), including altering one or more of the three CDRs of αCD3-VH or αCD3-VL, or any other functional variable domains. This can be achieved by: substituting one or more amino acid residues at functionally important residues in one or more CDRs, replacing some or all CDR residues with a random sequence, replacing one or more CDRs with a tag or label sequence, and / or exchanging CDRs and / or variable regions with those regions from unrelated antibodies (antibodies targeting proteins from different organisms, such as chicken egg lysozyme). In some cases, inactivation can be achieved by altering only one CDR in the variable region, but other embodiments also include altering one, two, three, four, five, or six CDRs.

[0307] The inactive GD of the present invention VL

[0308] In an example of the present invention, inactive GD VL Identification was performed by scanning the LCDR3 region of the CD3-specific antibody “αCD3-Fv” for alanine. The generation of αCD3-Fv is described in WO 2022 / 063819 (which is incorporated herein by reference in its entirety).

[0309] In the embodiments, according to the GD disclosed herein VL VL containing the following amino acid sequence: QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQ (SEQ ID NO: 14).

[0310] The inactive GD of the present invention VH

[0311] In this disclosure, there is no active GD VH Identification was performed by affinity maturation and deimmunization of αCD3-Fv, as described in unpublished European application EP 22163663.2 (which is incorporated herein by reference in its entirety).

[0312] In the embodiments, according to the GD disclosed herein VHVH containing the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWVRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 15).

[0313] Protected-Groove Variable Structural Domain (GD) VD-杵 )

[0314] In this disclosure, GD can be further modified. VD The germline framework region is designed to introduce protrusions into its interface region, with the aim of further reducing the ratio of heterodimer formation of two complementary CyCAT hemimolecules in solution and in the absence of target cells.

[0315] Those skilled in the art will understand that the engineered GD disclosed herein VD The "interface" includes GD VD Those contact amino acid residues in the antibody that interact with or will contact one or more contact amino acid residues at the interface of the complementary antibody variable domain.

[0316] As used in this article, "protrusion" refers to a bulge from GD VD The interface protrudes and thus destabilizes at least one amino acid side chain of the potential heterodimer. The protrusion is achieved by replacing the GD-derived amino acid side chain with a larger side chain (e.g., tyrosine (Y) or tryptophan (W)). VD The protrusions are constructed using small amino acid side chains at the interface. In some embodiments, the protrusions comprise substitutions to GD... VD The amino acid residues in the interface, wherein the substituted amino acid residues are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some embodiments, the amino acid residues with a side chain volume larger than that of the original amino acid residue are selected from R, F, Y, and W. In some embodiments, the original amino acid residues with a smaller side chain volume are selected from A, S, T, and V.

[0317] In some embodiments, at least one amino acid residue is replaced by an amino acid residue with a side chain volume larger than that of the original amino acid residue, thereby generating a protrusion within the interface, wherein the protrusion is located in the antibody variable heavy chain domain (VH).

[0318] In some embodiments, according to the Chothia annotation, GD VHThe amino acid residues at positions 37, 39, 44, 45, and / or 47 of the interface are substituted. In some embodiments, GD is replaced according to Chothia's notes. VH The amino acid residue at position 37 of the interface is replaced. In some embodiments, according to Kabat, GD VH The V at position 37 of the interface is replaced with R, F, Y, or W. In some embodiments, according to Kabat, GD VH The V at position 37 of the interface is replaced with F. In some embodiments, according to Kabat, GD having SEQ ID NO: 15 is replaced with F. VH The amino acid residue at position 37 of the interface is replaced.

[0319] In the embodiments, according to the GD disclosed herein VH-杵 VH containing the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 16).

[0320] In an embodiment, the GD VH According to this disclosure, GD VH-杵 .

[0321] HSA Protective Domain

[0322] The CyCAT half-molecule disclosed herein may include a half-life extension domain as a protective domain, such as domain 1, domain 2, or domain 3 of human serum albumin (HSA).

[0323] Human serum albumin (HSA; Uniprot: P02768) (molecular weight approximately 67 kDa) is the most abundant protein in human plasma and has a half-life of approximately 20 days in the human body. Non-covalent association with albumin prolongs the elimination half-life of short-lived proteins.

[0324] In the embodiments, the GD disclosed herein is or contains human serum albumin (GD). HSA-wt ) or fragments or portions or domains of human serum albumin (GD) HSA-frag. In embodiments, the GD according to this disclosure comprises an HSA or a truncated HSA. In embodiments, the GD according to this disclosure comprises an HSA having the following Uniprot: P02768 amino acid sequence (excluding the signal sequence):

[0325] (SEQ ID NO: 17).

[0326] In some embodiments, the hemimolecules described herein comprise truncated and / or variant forms of HSA, preferably as long as pH-sensitive binding with FcRn is retained. Binding to FcRn can be assessed by binding assays such as biolayer interferometry (BLI, Octet) or surface plasmon resonance (SPR, BIAcore).

[0327] In some embodiments, the truncated form of HSA comprises domain III (HSA-DIII) of HSA having the following amino acid sequence:

[0328] LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA (SEQ ID NO: 18)

[0329] Other suitable HSA truncations and HSA variants are known in the art. See, for example, US 10,711,050 and Sand et al., JBC [Journal of Biochemistry] 289(5):34583 (2014), both of which are incorporated herein by reference in their entirety.

[0330] In some embodiments, the GD according to this disclosure has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the GD according to this disclosure has the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the HSA is a variant HSA comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid modifications (e.g., substitution, addition and / or deletion).

[0331] The constructions and forms of this invention are variations or improvements of the inventions described in WO 2013 / 156178, WO 2022 / 248662, and WO 2023 / 006809, which are hereby expressly incorporated herein by reference in their entirety.

[0332] CyCAT half-molecule

[0333] According to this disclosure and as Figures 1-3The CyCAT half-molecules shown can freely combine into complementary half-molecule pairs, provided that one half carries at least one unpaired αCD3-VH domain and the other half carries at least one unpaired αCD3-VL domain.

[0334] Therefore, the possible complementary pairs of the half-molecules according to this disclosure are Figures 1-3 The following are some of the molecules or any other combination of molecules represented as A / A', B / B', C / C', D / D', E / E', F / F', such as A / B', A / C', A / D', A / E', A / F', B / A', B / C', B / D', B / E', B / F', C / A', C / B', C / D', C / E', C / F', D / A', D / B', D / C', D / E', D / F', E / A', E / B', E / C', E / D', E / F', F / A', F / B', F / C', F / D', F / E'.

[0335] CyCAT half lacking protected structural domain

[0336] In an embodiment, this disclosure provides a CyCAT half-molecule lacking the protective domains described herein.

[0337] In an embodiment, this disclosure relates to a half-molecule comprising

[0338] a) The first Fab, which is specific to the first antigen.

[0339] b) First peptide linker.

[0340] c) The first VH (αCD3-VH1) or first VL (αCD3-VL1) of Fv (αCD3-Fv) that is specific to CD3.

[0341] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of αCD3-VH1 or αCD3-VL1 via a first peptide linker.

[0342] In the embodiments, the structure of the half-molecule is as follows: Figure 1 A or Figure 1 As shown in A'.

[0343] In some embodiments, the hemimolecule according to this disclosure further comprises

[0344] d) Second peptide linker,

[0345] e) An Fc region composed of first and second Fc region subunits, wherein each Fc region subunit is composed of CH2 and CH3 domains.

[0346] f) The third peptide linker, and

[0347] The C-terminus of αCD3-VH1 or αCD3-VL1 is fused to the N-terminus of the first Fc region subunit via a second peptide linker, and the C-terminus of the third peptide linker is fused to the N-terminus of the second Fc region subunit.

[0348] In the embodiments, the structure of the half-molecule is as follows: Figure 2 A or Figure 2 As shown in A'.

[0349] In some embodiments, the hemimolecule according to this disclosure further comprises a second Fab that is specific to the first antigen, wherein the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the second Fc region subunit via a third peptide linker.

[0350] In the embodiments, the structure of the half-molecule is as follows: Figure 2 B or Figure 2 As shown in B'.

[0351] In alternative embodiments, the hemimolecule according to this disclosure further comprises

[0352] g) A second Fab that is specific to the first antigen.

[0353] h) Third peptide linker.

[0354] i) Second αCD3-VH1 or second αCD3-VL1,

[0355] j) The fourth peptide linker, and

[0356] The C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the second αCD3-VH1 or the second αCD3-VL1 via a third peptide linker, and the C-terminus of the second αCD3-VH1 or the second αCD3-VL1 is fused to the N-terminus of the second Fc subunit via a fourth peptide linker. If the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VH, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VH, or if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VL, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VL.

[0357] In the embodiments, the first αCD3-VH1 and the second αCD3-VH1 are identical. In the embodiments, the first αCD3-VL1 and the second αCD3-VL1 are identical. In the embodiments, the structure of the half-molecule is as follows: Figure 2 C or Figure 2 As shown in C'.

[0358] In some embodiments, this disclosure relates to a hemimolecule comprising

[0359] a) The first Fab, which is specific to the first antigen.

[0360] b) First peptide linker.

[0361] c) The Fc region composed of the first Fc region subunit and the second Fc region subunit.

[0362] d) Second peptide linker,

[0363] e) The first αCD3-VH1 or the first αCD3-VL1 of αCD3-Fv, and

[0364] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first Fc subunit via a first peptide linker, and the N-terminus of the first αCD3-VH1 or the first αCD3-VL1 is fused to the C-terminus of the first Fc subunit via a second peptide linker, respectively.

[0365] In the embodiments, the structure of the half-molecule is as follows: Figure 2 D or Figure 2 As shown in D'.

[0366] In some embodiments, the hemimolecule according to this disclosure further comprises

[0367] f) A second Fab that is specific to the first antigen.

[0368] g) Third peptide linker

[0369] The C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the second Fc subunit via a third peptide linker.

[0370] In the embodiments, the structure of the half-molecule is as follows: Figure 2 E or Figure 2 As shown in E'.

[0371] In alternative embodiments, the hemimolecule according to this disclosure further comprises a second αCD3-VH1 or a second αCD3-VL1, wherein the N-terminus of the second αCD3-VH1 or the second αCD3-VL1 is fused to the C-terminus of the second Fc region subunit via a fourth peptide linker, and wherein if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VH, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VH1, or if the first αCD3-VH1 or the first αCD3-VL1 is VL, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VL1.

[0372] In one embodiment, the first αCD3-VH1 and the second αCD3-VH1 are the same. In another embodiment, the first αCD3-VL1 and the second αCD3-VL1 are the same.

[0373] In the embodiments, the structure of the half-molecule is as follows: Figure 2 F or Figure 2 As shown in F'.

[0374] Complementary pairs of CyCAT halves lacking protected structural domains

[0375] In embodiments, this disclosure relates to a pair of complementary CyCAT hemimolecules, which contain

[0376] I. The first hemimolecule, which contains

[0377] a) The first Fab, which is specific to the first antigen.

[0378] b) First peptide linker.

[0379] c) The first VH (αCD3-VH1) or first VL (αCD3-VL1) of Fv that is specific to CD3.

[0380] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first αCD3-VH1 or the first αCD3-VL1 via a first peptide linker, and

[0381] II. The second hemimolecule, which contains

[0382] d) A second Fab that is specific to the second antigen.

[0383] e) Fifth peptide linker,

[0384] f) complementary first VH (αCD3-VH2) or complementary first VL (αCD3-VL2) of Fv that are specific to CD3, and

[0385] The C-terminus of the heavy chain of the second Fab is fused to the N-terminus of either the complementary first αCD3-VH2 or the complementary first αCD3-VL2 via a fifth peptide linker.

[0386] In embodiments, the first half-molecule according to this disclosure further comprises

[0387] g) Second peptide linker,

[0388] h) The first Fc region, composed of the first Fc region subunit and the second Fc region subunit.

[0389] i) Third peptide linker

[0390] The C-terminus of the first αCD3-VH1 or the first αCD3-VL1 is fused to the N-terminus of the first Fc region subunit via a second peptide linker, and the C-terminus of the third peptide linker is fused to the N-terminus of the second Fc region subunit.

[0391] In an embodiment, the first half-molecule according to this disclosure further comprises a third Fab that is specific to the first antigen, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second Fc region subunit via a third peptide linker.

[0392] In alternative embodiments, the first half-molecule according to this disclosure further comprises

[0393] g) The third Fab, which is specific to the first antigen,

[0394] h) Third peptide linker.

[0395] i) Second αCD3-VH1 or second αCD3-VL1,

[0396] j) The fourth peptide linker,

[0397] The C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second αCD3-VH1 or the second αCD3-VL1 via a third peptide linker, wherein the C-terminus of the second αCD3-VH1 or the second αCD3-VL1 is fused to the N-terminus of the second Fc subunit via a fourth peptide linker, and wherein if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VH, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VH, or if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VL, then the second αCD3-VH1 or the αCD3-VL1 is αCD3-VL.

[0398] In another embodiment, the second half-molecule according to this disclosure further comprises

[0399] g) The sixth peptide linker,

[0400] h) The second Fc region, composed of the third Fc region subunit and the fourth Fc region subunit.

[0401] i) The seventh peptide linker, and

[0402] The C-terminus of the complementary first αCD3-VH2 or the complementary first αCD3-VL2 is fused to the N-terminus of the third Fc subunit via a sixth peptide linker, and the C-terminus of the seventh peptide linker is fused to the N-terminus of the fourth Fc subunit.

[0403] In an embodiment, the second half-molecule according to this disclosure further comprises a fourth Fab that is specific to the second antigen, wherein the C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the second Fc region subunit via a seventh peptide linker.

[0404] In alternative embodiments, the second hemimolecule according to this disclosure further comprises

[0405] j) A fourth Fab that is specific to the second antigen,

[0406] k) Seventh peptide linker,

[0407] l) complementary second αCD3-VH2 or complementary second αCD3-VL2, and

[0408] m) The eighth peptide linker, and

[0409] The C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the complementary second αCD3-VH2 or the complementary second αCD3-VL2 via a seventh peptide linker, wherein the C-terminus of the complementary second αCD3-VH2 or the complementary second αCD3-VL2 is fused to the N-terminus of the fourth Fc subunit via an eighth peptide linker, wherein if the complementary first αCD3-VH2 or the complementary first αCD3-VL2 is the complementary first αCD3-VH2, then the complementary second αCD3-VH2 or the complementary second αCD3-VL2 is the complementary second αCD3-VH, and if the complementary first αCD3-VH2 or the complementary first αCD3-VL2 is the complementary first αCD3-VL2, then the complementary second αCD3-VH2 or the complementary second αCD3-VL2 is the complementary second αCD3-VL.

[0410] In an alternative embodiment, the second hemimolecule comprises

[0411] a) A second Fab that is specific to the second antigen.

[0412] b) Fifth peptide linker,

[0413] c) The second Fc region, composed of the third Fc region subunit and the fourth Fc region subunit.

[0414] d) The sixth peptide linker,

[0415] e) Seventh peptide linker

[0416] f) complementary first αCD3-VH2 or complementary first αCD3-VL2 of Fv that is specific to CD3, and

[0417] The N-terminus of the complementary first αCD3-VH2 or the complementary first αCD3-VL2 is fused to the C-terminus of the third Fc region subunit via a sixth peptide linker, and the C-terminus of the seventh peptide linker is fused to the N-terminus of the second Fc region.

[0418] In another embodiment, the second hemimolecule further comprises a fourth Fab that is specific to the second antigen, wherein the N-terminus of the fourth Fc subunit is fused to the C-terminus of the heavy chain of the second Fab via a seventh peptide linker.

[0419] In another embodiment, the second hemimolecule further comprises an eighth peptide linker and a complementary second αCD3-VH2 or a complementary second αCD3-VL2.

[0420] The N-terminus of the complementary second αCD3-VH2 or the complementary second αCD3-VL2 is fused to the C-terminus of the fourth Fc subunit via the eighth peptide linker.

[0421] CyCAT half-molecules containing protective domains

[0422] In an embodiment, this disclosure relates to a half-molecule comprising:

[0423] a) The first Fab, which is specific to the first antigen.

[0424] b) First peptide linker.

[0425] c) The first VH (αCD3-VH) or first VL (αCD3-VL) of Fv (αCD3-Fv) that is specific to CD3.

[0426] d) Second peptide linker,

[0427] e) The first Fc region is composed of the first Fc region subunit and the second Fc region subunit.

[0428] f) Third peptide linker

[0429] g) First protective structural domain (GD), and

[0430] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first αCD3-VH or the first αCD3-VL via a first peptide linker, and the C-terminus of the first αCD3-VH or the first αCD3-VL is fused to the N-terminus of the first Fc subunit via a second peptide linker, and the C-terminus of GD is fused to the N-terminus of the second Fc subunit via a third peptide linker.

[0431] In the embodiments, the structure of the half-molecule is as follows: Figure 2 A or Figure 2 As shown in A'.

[0432] In the embodiments, the half-molecule further comprises

[0433] h) The second Fab and fourth peptide linkers are specific to the first antigen.

[0434] The C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the GD via a fourth peptide linker.

[0435] In the embodiments, the structure of the half-molecule is as follows: Figure 2 C or Figure 2 As shown in C'.

[0436] In an alternative embodiment, this disclosure relates to a half-molecule comprising:

[0437] a) The first Fab, which is specific to the first antigen.

[0438] b) First peptide linker.

[0439] c) First protective structural domain.

[0440] d) Second peptide linker,

[0441] e) The first Fc region is composed of the first Fc region subunit and the second Fc region subunit.

[0442] f) Third peptide linker

[0443] g) The first VH (αCD3-VH1) or first VL (αCD3-VL1) of Fv (αCD3-Fv) that is specific to CD3, and

[0444] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first GD via a first peptide linker, the C-terminus of the GD is fused to the N-terminus of the first Fc subunit via a second peptide linker, and the C-terminus of the first αCD3-VH1 or the first αCD3-VL1 is fused to the N-terminus of the second Fc subunit via a third peptide linker.

[0445] In the embodiments, the structure of the half-molecule is as follows: Figure 2 B or Figure 2 As shown in B'.

[0446] In an alternative embodiment, this disclosure relates to a half-molecule comprising

[0447] a) The first Fab, which is specific to the first antigen.

[0448] b) First peptide linker.

[0449] c) The first Fc region, composed of the first Fc region subunit and the second Fc region subunit.

[0450] d) Second peptide linker,

[0451] e) The first VH (αCD3-VH1) or first VL (αCD3-VL1) of Fv (αCD3-Fv) that is specific to CD3.

[0452] f) Third peptide linker

[0453] g) Fourth peptide linker,

[0454] h) Protective structural domain (GD), and

[0455] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first Fc subunit via a first peptide linker, the N-terminus of the first αCD3-VH1 or the first αCD3-VL1 is fused to the C-terminus of the first Fc subunit via a second peptide linker, the N-terminus of GD is fused to the C-terminus of the second Fc subunit via a third peptide linker, and the N-terminus of the second Fc subunit is fused to a fourth peptide linker.

[0456] In the embodiments, the structure of the molecule is as follows: Figure 2 D、 Figure 2 As shown in D'.

[0457] In another embodiment, the hemimolecule further comprises a second Fab that is specific to the first antigen, wherein the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the second Fc region subunit via a fourth peptide linker.

[0458] In the embodiments, the structure of the half-molecule is as shown in 3E or 3E'.

[0459] All contain complementary pairs of CyCAT halves that protect the structural domain.

[0460] In embodiments, this disclosure relates to a pair of complementary half-molecules, which contain

[0461] I) The first half-molecule, which contains

[0462] a) The first Fab, which is specific to the first antigen.

[0463] b) First peptide linker.

[0464] c) The first αCD3-VH1 or the first αCD3-VL1 of αCD3-Fv

[0465] d) Second peptide linker,

[0466] e) The Fc region composed of the first Fc region subunit and the second Fc region subunit.

[0467] f) Third peptide linker

[0468] g) First protective structural domain (GD), and

[0469] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first αCD3-VH1 or the first αCD3-VL1 via a first peptide linker, wherein the C-terminus of the first αCD3-VH1 or the first αCD3-VL1 is fused to the N-terminus of the first Fc subunit via a second peptide linker, and wherein the C-terminus of the first GD is fused to the N-terminus of the second Fc subunit via a third peptide linker; and

[0470] II) The second hemimolecule, which contains

[0471] a) A second Fab that is specific to the second antigen.

[0472] b) Fifth peptide linker,

[0473] c) The complementary first αCD3-VH2 or complementary first αCD3-VL2 of αCD3-Fv

[0474] d) The sixth peptide linker, and

[0475] e) The second Fc region, composed of the third Fc region subunit and the fourth Fc region subunit.

[0476] f) Seventh peptide linker

[0477] g) Second protective structural domain (GD)

[0478] The C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the complementary first αCD3-VH2 or the complementary first αCD3-VL2 via a fifth peptide linker, the C-terminus of the complementary first αCD3-VH2 or the complementary first αCD3-VL2 is fused to the N-terminus of the third Fc subunit via a sixth peptide linker, and the C-terminus of the second GD is fused to the N-terminus of the fourth Fc subunit via a seventh peptide linker.

[0479] In another embodiment, the first hemimolecule further comprises a third Fab and a fourth peptide linker that are specific to the first antigen, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the first GD via the fourth peptide linker.

[0480] In an embodiment, the second hemimolecule further comprises a fourth Fab and an eighth peptide linker that are specific to the second antigen, wherein the C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the second GD via the eighth peptide linker.

[0481] In alternative embodiments, this disclosure relates to a pair of hemimolecules comprising:

[0482] I) The first half-molecule, which contains

[0483] a) The first Fab, which is specific to the first antigen.

[0484] b) First peptide linker.

[0485] c) First protective structural domain (GD).

[0486] d) Second peptide linker,

[0487] e) The first Fc region is composed of the first Fc region subunit and the second Fc region subunit.

[0488] f) The third peptide linker, and

[0489] g) First αCD3-VH1 or first αCD3-VL1,

[0490] The C-terminus of the first Fab heavy chain is fused to the N-terminus of the first GD via a first peptide linker, the C-terminus of the first GD is fused to the N-terminus of the first Fc subunit via a second peptide linker, and the C-terminus of the first αCD3-VH1 or the first αCD3-VL1 is fused to the N-terminus of the second Fc subunit via a third peptide linker; and

[0491] II) The second hemimolecule, which contains

[0492] a) A second Fab that is specific to the second antigen.

[0493] b) The fourth peptide linker,

[0494] c) Second protective structural domain (GD)

[0495] d) Fifth peptide linker,

[0496] e) The second Fc region, composed of the third Fc region subunit and the fourth Fc region subunit.

[0497] f) The sixth peptide linker, and

[0498] g) Complementary first αCD3-VH2 or complementary first αCD3-VL2

[0499] The C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the second GD via a fourth peptide linker, the C-terminus of the second GD is fused to the N-terminus of the third Fc subunit via a fifth peptide linker, and the C-terminus of the complementary first αCD3-VH2 or the complementary first αCD3-VH2 is fused to the N-terminus of the fourth Fc subunit via a sixth peptide linker.

[0500] In embodiments, this disclosure relates to a pair of hemimolecules, which contain

[0501] I) The first half-molecule, which contains

[0502] a) Contains a first Fab that is specific to the first antigen.

[0503] b) First peptide linker.

[0504] c) The first Fc region, composed of the first Fc region subunit and the second Fc region subunit.

[0505] d) Second peptide linker,

[0506] e) The first αCD3-VH1 or the first αCD3-VL1 of αCD3-Fv

[0507] f) Third peptide linker

[0508] g) The fourth peptide linker, and

[0509] h) First protective structural domain (GD), and

[0510] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first Fc subunit via a first peptide linker; the N-terminus of the first αCD3-VH1 or the first αCD3-VL1 is fused to the C-terminus of the first Fc subunit via a second peptide linker; the N-terminus of the first GD is fused to the C-terminus of the second Fc subunit via a third peptide linker; and the N-terminus of the second Fc subunit is fused to a fourth peptide linker.

[0511] II) The second hemimolecule, which contains

[0512] a) Contains a second Fab that is specific to the second antigen.

[0513] b) Fifth peptide linker,

[0514] c) The second Fc region, composed of the third Fc region subunit and the fourth Fc region subunit.

[0515] d) The sixth peptide linker,

[0516] e) The complementary first αCD3-VH2 or complementary first αCD3-VL2 of αCD3-Fv

[0517] f) Seventh peptide linker

[0518] g) The eighth peptide linker, and

[0519] h) Second protective structural domain (GD2).

[0520] The C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the third Fc subunit via a fifth peptide linker, the N-terminus of the complementary first αCD3-VH2 or the complementary first αCD3-VL2 is fused to the C-terminus of the third Fc subunit via a second peptide linker, the N-terminus of the second GD is fused to the C-terminus of the fourth Fc subunit via a seventh peptide linker, and the N-terminus of the fourth Fc subunit is fused to an eighth peptide linker.

[0521] In some embodiments, the first hemimolecule further comprises a third Fab that is specific to the first antigen, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second Fc region subunit via a fourth peptide linker.

[0522] In some embodiments, the second hemimolecule further comprises a fourth Fab that is specific to the second antigen, wherein the C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the fourth Fc region subunit via an eighth peptide linker.

[0523] One of the halves contains a complementary pair of CyCAT halves that protect the structural domain.

[0524] In embodiments, this disclosure relates to a pair of hemimolecules, which contain

[0525] I) The first half-molecule, which contains

[0526] a) The first Fab, which is specific to the first antigen.

[0527] b) First peptide linker.

[0528] c) The first VH (αCD3-VH1) or the first VL (αCD3-VL1) of αCD3-Fv, and

[0529] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first αCD3-VH1 or the first αCD3-VL1 via a first peptide linker; and

[0530] II) The second hemimolecule, which contains

[0531] a) A second Fab that is specific to the second antigen.

[0532] b) Fifth peptide linker,

[0533] c) The first complementary VH (αCD3-VH2) or the first complementary VL (αCD3-VL2) of αCD3-Fv.

[0534] d) The sixth peptide linker,

[0535] e) The second Fc region, composed of the third Fc region subunit and the fourth Fc region subunit.

[0536] f) The seventh peptide linker, and

[0537] g) First protective structural domain (GD).

[0538] The C-terminus of the second Fab heavy chain is fused to the N-terminus of the first αCD3-VH2 or the first αCD3-VL via a fifth peptide linker, the C-terminus of the first αCD3-VH2 or the first αCD3-VL2 is fused to the N-terminus of the third Fc subunit via a sixth peptide linker, and the C-terminus of the first protecting domain is fused to the N-terminus of the second Fc subunit via a seventh peptide linker.

[0539] In some embodiments, the first hemimolecule further comprises

[0540] a) Second peptide linker,

[0541] b) The first Fc region, composed of the first Fc region subunit and the second Fc region subunit.

[0542] c) Third peptide linker

[0543] The C-terminus of the first αCD3-VH1 or the first αCD3-VL1 is fused to the N-terminus of the first Fc region subunit via a second peptide linker, and the C-terminus of the third peptide linker is fused to the N-terminus of the second Fc region subunit.

[0544] In some embodiments, the first half-molecule according to this disclosure further comprises a third Fab that is specific to the first antigen, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second Fc region subunit via a third peptide linker.

[0545] In alternative embodiments, the first half-molecule according to this disclosure further comprises a second αCD3-VH1 or a second αCD3-VL1 and a fourth peptide linker, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second αCD3-VH1 or the second αCD3-VL1 via the fourth peptide linker, wherein the C-terminus of the second αCD3-VH1 or the second αCD3-VL1 is fused to the N-terminus of the second Fc region subunit via the third peptide linker, and wherein if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VH, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VH, or if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VL, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VL.

[0546] In the embodiments disclosed herein, the first αCD3-VH1 and the second αCD3-VH1 or the first αCD3-VL1 and the second αCD3-VL1 are the same.

[0547] In some embodiments, the second hemimolecule further comprises a fourth Fab and an eighth peptide linker that are specific to the second antigen, and wherein the C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the first protective domain via the eighth peptide linker.

[0548] In embodiments, this disclosure relates to a pair of hemimolecules, which contain

[0549] I) The first half-molecule, which contains

[0550] a) The first Fab, which is specific to the first antigen.

[0551] b) First peptide linker.

[0552] c) The first VH (αCD3-VH1) or the first VL (αCD3-VL1) of αCD3-Fv, and

[0553] The C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first αCD3-VH1 or the first αCD3-VL1 via a first peptide linker; and

[0554] II) The second hemimolecule, which contains

[0555] a) A second Fab that is specific to the second antigen.

[0556] b) Fifth peptide linker,

[0557] c) First protective structural domain.

[0558] d) The sixth peptide linker,

[0559] e) The second Fc region, composed of the third Fc region subunit and the fourth Fc region subunit.

[0560] f) The seventh peptide linker, and

[0561] g) The complementary first VH (αCD3-VH2) or complementary first VL (αCD3-VL2) of αCD3-Fv.

[0562] The C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the first protective domain via a fifth peptide linker, the C-terminus of the first protective domain is fused to the N-terminus of the third Fc subunit via a sixth peptide linker, and the C-terminus of the first αCD3-VH2 or the first αCD3-VH2 is fused to the N-terminus of the fourth Fc subunit via a seventh peptide linker.

[0563] In some embodiments, the first hemimolecule further comprises

[0564] a) Second peptide linker, and

[0565] b) The first Fc region, composed of the first Fc region subunit and the second Fc region subunit, and

[0566] c) A third peptide linker, wherein the C-terminus of the first αCD3-VH1 or the first αCD3-VL1 is fused to the N-terminus of the first Fc region subunit via a second peptide linker, and wherein the C-terminus of the third peptide linker is fused to the N-terminus of the second Fc region subunit.

[0567] In some embodiments, the first hemimolecule further comprises a third Fab that is specific to the first antigen, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second Fc region subunit via a third peptide linker.

[0568] In an alternative embodiment, the first hemimolecule further comprises

[0569] d) The third Fab, which is specific to the first antigen,

[0570] f) Second αCD3-VH1 or second αCD3-VL1,

[0571] g) The fourth peptide linker, and

[0572] The C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second αCD3-VH1 or the second αCD3-VL1 via a third peptide linker, wherein the C-terminus of the second αCD3-VH1 or the second αCD3-VL1 is fused to the N-terminus of the second Fc subunit via a fourth peptide linker, wherein if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VH, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VH, or if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VL, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VL.

[0573] In the embodiments disclosed herein, the first αCD3-VH1 and the second αCD3-VH1 are the same, or the first αCD3-VL1 and the second αCD3-VL1 are the same.

[0574] In an alternative embodiment, the first hemimolecule comprises

[0575] a) The first Fab, which is specific to the first antigen.

[0576] b) First peptide linker.

[0577] c) The first Fc region, composed of the first Fc region subunit and the second Fc region subunit.

[0578] d) Second peptide linker,

[0579] e) Third peptide linker

[0580] f) The first αCD3-VH1 or the first αCD3-VL1 of αCD3-Fv

[0581] The N-terminus of αCD3-VH1 or αCD3-VL1 is fused to the C-terminus of the first Fc subunit via a second peptide linker, and the C-terminus of the third peptide linker is fused to the N-terminus of the second Fc subunit.

[0582] In some embodiments, the first hemimolecule further comprises a third Fab that is specific to the first antigen, wherein the N-terminus of the second Fc region subunit is fused to the C-terminus of the heavy chain of the third Fab via a third peptide linker.

[0583] In some embodiments, the first hemimolecule further comprises a fourth peptide linker and a second αCD3-VH1 or a second αCD3-VL1, wherein the N-terminus of the second αCD3-VH1 or the second αCD3-VL1 is fused to the C-terminus of a third Fc region subunit via the fourth peptide linker, and wherein if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VH, then the second αCD3-VH1 or the second αCD3-VL1 is αCD3-VH, or if the first αCD3-VH1 or the first αCD3-VL1 is αCD3-VL, then the second αCD3-VH1 or the αCD3-VL1 is αCD3-VL.

[0584] In the embodiments disclosed herein, the first αCD3-VH1 and the second αCD3-VH1 are the same, or the first αCD3-VL1 and the second αCD3-VL1 are the same.

[0585] peptide linkers

[0586] The CyCAT hemimolecule disclosed herein can be designed such that its components (e.g., targeting Fab fragments or unpaired αCD3-SVDs) fuse directly or indirectly with each other. In embodiments, the linker is a peptide linker. In some embodiments, the components of the CyCAT hemimolecule fuse with each other genetically. Such fusion can be achieved through a variety of strategies, including but not limited to peptide or polypeptide fusion between the N-terminus and C-terminus, fusion via disulfide bonds, and fusion via chemical crosslinking agents.

[0587] In the embodiments disclosed herein, the linker is a peptide linker comprising one or more amino acid residues linked by peptide bonds known in the art. The composition and length of the peptide linker can be determined according to methods well known in the art, and efficacy testing can be performed.

[0588] Peptide linkers should have an appropriate length to fuse two components so that they present the correct conformation to each other, thereby preserving or acquiring the desired activity or functionality.

[0589] In the embodiments, the peptide linkers according to this disclosure consist only of naturally occurring amino acid residues. In the embodiments, the peptide linkers are non-immunogenic. In the embodiments, the peptide linkers are unstructured. In the embodiments, the peptide linkers are flexible. In the embodiments, the peptide linkers do not contain protease cleavage sites.

[0590] In the embodiments, the peptide linker may comprise a GA polymer, an AS polymer, a PA polymer, or a PAS polymer, such as (GS). n (SEQ ID NO: 23), (G4S) n(SEQ ID NO: 24), (SG4) n (SEQ ID NO: 25), (GSGGS) n (SEQ ID NO: 26), (GGGS) n (SEQ ID NO: 27), G4 (SG4) n (SEQ ID NO: 28), (GGSG) n (SEQ ID NO: 29), (GGSGG) n (SEQ ID NO: 30), (GSGSG) n (SEQ ID NO: 31), (GSGGG) n (SEQ ID NO:32), (GGGSG) n (SEQ ID NO: 33) and (GSSSG) n (SEQ ID NO: 34), where n is an integer between 1 and 10, typically an integer between 2 and 4.

[0591] Suitable peptide linkers may also originate from constant domains of the immunoglobulin light or heavy chains, such as the CLκ or CLλ domain or the CH1 domain, but not all residues of such constant domains, for example only the first 5-12 amino acid residues. In the embodiments, the peptide linkers according to this disclosure comprise the following amino acid sequence: QPKAAP (SEQ ID NO: 35) or ASTKGP (SEQ ID NO: 36).

[0592] The peptide linker may also comprise an immunoglobulin hinge (e.g., a human IgG1 hinge or a portion thereof) or any peptide derived from such a hinge. Preferably, when only a portion or part of an immunoglobulin hinge is used, the truncated hinge may still include one or more of its interchain cysteine ​​residues. In the case of using two such hinged peptide linkers in two adjacent polypeptides, the presence of the interchain cysteine ​​allows for the formation of a dimer peptide linker (or hinge region) via a disulfide bridge. The presence of the dimer peptide linker or hinge region further promotes and stabilizes the dimerization of the two Fc region subunits that may be present in the CyCAT hemimolecule according to this disclosure.

[0593] In embodiments, the peptide linker according to this disclosure comprises an amino acid sequence selected from the group consisting of: EPKSCDKTHTCPPCP (SEQ ID NO: 37), DKTHTCPPCP (SEQ ID NO: 38), KTHTCPPCP (SEQ ID NO: 39), and KTHT (SEQ ID NO: 40). In other embodiments, the peptide linker according to this disclosure comprises an amino acid sequence selected from the group consisting of: QPKAAPDKTHTCPPCP (SEQ ID NO: 41); ASTKGPDKTHTCPPCP (SEQ ID NO: 42), QPKAAPKTHTCPPCP (SEQ ID NO: 43), or ASTKGPKTHTCPPCP (SEQ ID NO: 44).

[0594] In embodiments, the peptide linker according to this disclosure is composed of amino acid residues A, Q, D, P, H, G, S, E, T, K, and C. In embodiments, the peptide linker according to this disclosure is composed of amino acid residues selected from the group consisting of A, Q, D, P, H, and G. In embodiments, the peptide linker according to this disclosure comprises an amino acid sequence selected from the group consisting of: GQPSG (SEQ ID NO: 45), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 46), PAAPAP (SEQ ID NO: 47), PAAPAPDA (SEQ ID NO: 48), AQPAAPAPAE (SEQ ID NO: 49), DQPAAPAPDA (SEQ ID NO: 50), AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 51), DQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 52), AQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 53), DQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 54), AQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 45), GQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 46), GQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 47), GQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 48), GQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 49 ... 55), DQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 56), DQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 57), and AQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 58).

[0595] In some embodiments, the length of the peptide linker according to this disclosure is about 4 to 100, 4 to 50, or 4 to 30 amino acid residues. In some embodiments, the peptide linker comprises 1 to 200, 1 to 100, 1 to 70, 1 to 65, 1 to 50, 1 to 25, or 1 to 20 amino acids. In some embodiments, the length of the peptide linker is at least 4, at least 5, at least 10, at least 15, or at least 20 amino acid residues. In some embodiments, the length of the peptide linker is no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, or no more than 100 amino acid residues. In the embodiments, the peptide linker according to this disclosure has a length between 5 and 50 amino acid residues, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues. In the embodiments, the peptide linker has a length of 5 to 50 amino acid residues. In the embodiments, the length of the peptide linker according to this disclosure is 5 to 50 amino acid residues, 5 to 45 amino acid residues, 5 to 40 amino acid residues, 5 to 35 amino acid residues, 5 to 30 amino acid residues, 5 to 25 amino acid residues, 5 to 20 amino acid residues, 5 to 15 amino acid residues, or 5 to 10 amino acid residues. In the embodiments, the length of the peptide linker is 5 to 40 amino acid residues. In the embodiments, the length of the peptide linker is 9 to 29 amino acid residues. In the embodiments, the length of the peptide linker is 5 to 29 amino acid residues. In the embodiments, the length of the peptide linker is 5 amino acid residues, 8 amino acid residues, 9 amino acid residues, 10 amino acid residues, 15 amino acid residues, 20 amino acid residues, 29 amino acid residues, 40 amino acid residues, or 49 amino acid residues.

[0596] In one embodiment, the peptide linker is 5 amino acid residues long. In another embodiment, the peptide linker is 8 amino acid residues long. In another embodiment, the peptide linker is 9 amino acid residues long. In another embodiment, the peptide linker is 10 amino acid residues long. In another embodiment, the peptide linker is 15 amino acid residues long. In another embodiment, the peptide linker is 19 amino acid residues long. In another embodiment, the peptide linker is 20 amino acid residues long. In another embodiment, the peptide linker is 25 amino acid residues long. In another embodiment, the peptide linker is 29 amino acid residues long. In another embodiment, the peptide linker is 40 amino acid residues long. In another embodiment, the peptide linker is 45 amino acid residues long. In another embodiment, the peptide linker is 49 amino acid residues long.

[0597] It should be understood that, as used herein, peptide linkers are not limited to only one of the foregoing and exemplary peptide linkers, but may include any combination of two or more such linkers fused together. For example, as used herein, peptide linkers may be constructed from GS polymers and immunoglobulin hinge-derived sequences. In embodiments, as used herein, the peptide linker does not contain an IgG Fc region. In embodiments, the peptide linker does not contain monomers of IgG Fc region subunits. In embodiments, the peptide linkers present in the CyCAT hemimolecule according to this disclosure are identical. In embodiments, the peptide linkers are different. In embodiments, the peptide linkers have the same length. In embodiments, the peptide linkers have different lengths.

[0598] Preferred peptide linkers

[0599] Preferably, the protective domain according to this disclosure is fused to the half-molecule in an "indivisible" manner.

[0600] "Uncleavable" means that the CyCAT hemimolecule cannot be cleaved and released by the activity of enzymes expressed by cancer cells, and such CyCAT hemimolecules bind to cancer cells. For example, the GD disclosed herein can fuse with the hemimolecule via peptide linkers (specifically, via uncleavable peptide linkers).

[0601] In this document, "uncleanable peptide linker" refers to an amino acid sequence that cannot be cleaved by human proteases under normal physiological conditions. Therefore, the GD and / or peptide linkers fused with GD to hemimolecules according to this disclosure do not contain protease cleavage sites. Therefore, the GD and / or peptide linkers according to this disclosure cannot be cleaved by proteases. In embodiments, the protease comprises a tumor-specific protease. In embodiments, the protease comprises a matrix metalloproteinase (MMP) or a serine protease. In embodiments, the matrix metalloproteinase comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In embodiments, the serine protease comprises a matriptase, urokinase, or hepsin.

[0602] As indicated herein, a “protease cleavage site” is an amino acid sequence that can be cleaved by a protease (such as matrix metalloproteinase or furin). Examples of such sites include: Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln (SEQ ID NO: 59) or Ala-Val-Arg-Trp-Leu-Leu-Thr-Ala (SEQ ID NO: 60) (cleaved by metalloproteinases), or Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 61) (cleaved by furin).

[0603] In therapeutic applications, protease cleavage sites can be cleaved by proteases produced by target cells (e.g., cancer cells, infected cells, or pathogens). Cancer cells are known to express proteases such as matrix metalloproteinases, and cancer-associated proteases and their corresponding cleavage sites (i.e., the specific amino acid sequences recognized by such proteases) are well known in the art. Cancer-associated protease families can include metalloproteinases, serine proteases, cysteine ​​proteases, aspartic proteases, and threonine proteases. Protease databases include PMAP (… Figure 2 The databases available for identification include .org, ExPASy PeptideCutter (ca.expasy.org / tools / peptidecutter), and PMAP.Cut DB (cutdb.burnham.org). Other databases that help identify specific protease cleavage sites include Oncomine (www.oncomine.org), the European Institute for Bioinformatics (www.ebi.ac.uk), and especially (www.ebi.ac.uk / gxa).

[0604] In embodiments, the GD according to this disclosure does not contain a protease cleavage site. In embodiments, the GD according to this disclosure does not contain a protease cleavage site, particularly a cancer-associated protease cleavage site. In embodiments, the peptide linker for fusing GD to a CyCAT hemimolecule according to this disclosure does not contain a protease cleavage site. In embodiments, the peptide linker for fusing GD to a CyCAT hemimolecule according to this disclosure does not contain a protease cleavage site, particularly a protease cleavage site that is cleaved by or cleavable by cancer-associated proteases. In one such embodiment, GD is fused to an Fc subunit of a CyCAT hemimolecule. In embodiments, GD according to this disclosure is not released from the CyCAT hemimolecule. In embodiments, the GD domain is not released from the CyCAT hemimolecule via proteolytic cleavage.

[0605] In some embodiments, the GD domain according to this disclosure is not released from the half-molecule after administration to a subject. In some embodiments, the GD domain is not released from the half-molecule by proteolytic cleavage after administration to a subject. In some embodiments, the GD domain is not released from the half-molecule by proteolytic cleavage of a protease after administration to a subject. In some embodiments, the GD domain is not released from the half-molecule by proteolytic cleavage of a cancer-associated protease after administration to a subject.

[0606] Antibody

[0607] The antibodies or antibody fragments used in the CyCAT hemimolecule disclosed herein, as well as the VH and VL domains, can be of any animal species origin, such as mice, rats, humans, or non-human primates. Preferably, the source is human or can also be obtained through humanization methods.

[0608] CD3-binding domain

[0609] Many suitable CD3-specific antibodies and / or corresponding VH and VL domains or CDRs known in the art are available for use in this disclosure.

[0610] For example, the CDR and / or VH and VL domains are derived from known anti-CD3 antibodies, such as moromuzumab-CD3 (OKT3), oxizumab (TRX4), telizumab (MGA031), vexizumab (Nuvion), SP34 or any humanized variant of SP34, I2C, H2C, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII- 87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

[0611] Preferably, the VH and VL domains forming the active CD3-specific Fv fragment are those described in International Application No. PCT / EP2021 / 076052 (which is incorporated herein by reference in its entirety). In embodiments of this disclosure, the VH and VL of the CD3-specific Fv according to this disclosure are derived from any of the CD3-specific antibodies disclosed in International Application No. PCT / EP2021 / 076052, or their derivatives or variants. In embodiments, the αCD3-Fv according to this disclosure competes with CD3-specific antibodies for binding to epitopes on CD3 (particularly CD3ε). In embodiments, the αCD3-Fv according to this disclosure competes with any of the CD3-specific antibodies disclosed herein. In embodiments, the αCD3-Fv according to this disclosure competes with any of the CD3-specific antibodies disclosed in International Application No. PCT / EP2021 / 076052. In the embodiments disclosed herein, αCD3-Fv includes any VH and / or VL domains disclosed in PCT / EP2021 / 076052. In the embodiments, αCD3-Fv competes with any of the CD3-specific antibodies disclosed in Table 2 or Table 3 of this specification for binding to an epitope on CD3.

[0612] In an embodiment, αCD3-Fv according to the present disclosure competes with antibodies comprising αCD3-VH having an amino acid sequence having SEQ ID NO: 7 or SEQ ID: 12 and αCD3-VL having an amino acid sequence having SEQ ID NO: 8 or SEQ ID NO: 13 for binding to an epitope on CD3.

[0613] In the embodiments, the VH and VL domains and / or their corresponding CDR regions incorporated into the CyCAT half-molecule according to this disclosure can form an active anti-CD3 Fv domain (αCD3-Fv) that binds to human CD3, as shown in Tables 2 and 3 of this specification.

[0614] Table 2: VH, VL, and CDR amino acid sequences of low-affinity αCD3ε antibodies that can be used in the half-molecules disclosed herein.

[0615]

[0616] Table 3: VH, VL, and CDR amino acid sequences of high-affinity αCD3ε antibodies that can be used in the half-molecules disclosed herein.

[0617]

[0618] In an embodiment, the αCD3-VH and αCD3-VL domains (collectively referred to as “split variable domains” or “αCD3-SVD”) form an active anti-CD3 Fv domain (αCD3-Fv). In an embodiment, the αCD3-Fv is composed of the αCD3-VL and αCD3-VH domains as disclosed herein. In an embodiment, the αCD3-VH and αCD3-VL are complementary to each other. In an embodiment, the αCD3-VH and αCD3-VL are capable of non-covalent dimerization with each other. In an embodiment, the αCD3-VH and αCD3-VL are capable of non-covalent association with each other. In an embodiment, the non-covalent association leads to the formation of αCD3-Fv. In an embodiment, the dimerization or non-covalent association occurs in solution. In an embodiment, the dimerization or non-covalent association occurs in solution in the presence of target cells. In the embodiments disclosed herein, the αCD3-VH and the αCD3-VL are not covalently linked. In the embodiments disclosed herein, the αCD3-VH itself cannot bind to CD3. In the embodiments disclosed herein, the αCD3-VL itself cannot bind to CD3. In the embodiments disclosed herein, neither the αCD3-VH nor the αCD3-VL of αCD3-Fv itself can bind to CD3.

[0619] In some embodiments, the αCD3-Fv according to this disclosure binds to human CD3. In some embodiments, the αCD3-Fv according to this disclosure binds to both human CD3 and cynomolgus monkey CD3. In some embodiments, the αCD3-Fv according to this disclosure binds to human CD3 and also cross-reactively binds to cynomolgus monkey CD3. In some embodiments, the binding of the αCD3-Fv to CD3 is specific.

[0620] In some embodiments, αCD3-Fv binds to immune cells expressing CD3. In some embodiments, αCD3-Fv binds to T cells expressing CD3. In some embodiments, αCD3-Fv binds to cytotoxic T cells expressing CD3. In some embodiments, αCD3-Fv binds to human cells expressing human CD3. In some embodiments, αCD3-Fv binds to cynomolgus monkey cells expressing cynomolgus monkey CD3. In some embodiments, αCD3-Fv binds to both human cells expressing human CD3 and cynomolgus monkey cells expressing cynomolgus monkey CD3.

[0621] In the embodiments disclosed herein, CD3 is CD3ε. In the embodiments, CD3 is human CD3ε. In the embodiments, human CD3ε comprises the amino acid sequence of SEQ ID NO: 19. In the embodiments, CD3 is cynomolgus monkey CD3ε. In the embodiments, cynomolgus monkey CD3ε comprises the amino acid sequence of SEQ ID NO: 21. In the embodiments, αCD3-Fv according to the present disclosure binds to the extracellular region of CD3ε. In the embodiments, αCD3-Fv binds to the extracellular region of human CD3ε. In the embodiments, αCD3-Fv binds to the extracellular region of cynomolgus monkey CD3ε. In the embodiments, αCD3-Fv binds to the extracellular region of both human and cynomolgus monkey CD3ε. In the embodiments, the extracellular region of human CD3ε comprises the amino acid sequence of SEQ ID NO: 20. In the embodiments, the extracellular region of cynomolgus monkey CD3ε comprises the amino acid sequence of SEQ ID NO: 22. In the embodiments, αCD3-Fv binds to a human CD3ε polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20.

[0622] In an embodiment, αCD3-Fv according to the present disclosure binds to a cynomolgus CD3ε polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22. In another embodiment, αCD3-Fv according to the present disclosure specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0623] In an embodiment, the αCD3-VH according to this disclosure comprises an HCDR1 region containing an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, an HCDR2 region containing an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and an HCDR3 region containing an amino acid sequence of SEQ ID NO: 3.

[0624] In the embodiments disclosed herein, αCD3-VH comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to the group consisting of SEQ ID NO: 7 and SEQ ID NO: 12. In the embodiments disclosed herein, αCD3-VH comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 12.

[0625] In embodiments, the αCD3-VL according to this disclosure comprises an LCDR1 region containing the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 11, an LCDR2 region containing the amino acid sequence of SEQ ID NO: 5, and an LCDR3 region containing the amino acid sequence of SEQ ID NO: 6. In embodiments of this disclosure, αCD3-VL comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13. In embodiments of this disclosure, αCD3-VL comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13.

[0626] In an embodiment, αCD3-Fv according to this disclosure includes

[0627] a) αCD3-VH, comprising an HCDR1 region containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, an HCDR2 region containing the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and an HCDR3 region containing the amino acid sequence of SEQ ID NO: 3, and

[0628] b) αCD3-VL, comprising an LCDR1 region containing the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 11, an LCDR2 region containing the amino acid sequence of SEQ ID NO: 5, and an LCDR3 region containing the amino acid sequence of SEQ ID NO: 6.

[0629] In the embodiments disclosed herein, αCD3-Fv comprises αCD3-VH and αCD3-VL, wherein αCD3-VH comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 12, and αCD3-VL comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13.

[0630] In the embodiments disclosed herein, αCD3-Fv comprises αCD3-VH and αCD3-VL, wherein αCD3-VH comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 12, and αCD3-VL comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13.

[0631] In the embodiments, αCD3-Fv according to this disclosure is an isolated antibody fragment. In the embodiments, αCD3-Fv is a human antibody fragment. In the embodiments, αCD3-Fv is a recombinant antibody fragment. In the embodiments, αCD3-Fv is a synthetic antibody fragment. In the embodiments, αCD3-Fv is a chimeric antibody fragment. In the embodiments, αCD3-Fv is a monoclonal antibody fragment.

[0632] Fc area

[0633] The Fc region of the CyCAT hemimolecule may contain or consist of a pair of polypeptides containing heavy chain constant domains of conventional immunoglobulins. The Fc region of conventional IgG exists as a dimer, with each subunit containing CH2 and CH3 IgG heavy chain constant domains. The two Fc region subunits are capable of stably associating with each other. Therefore, in the embodiments, the two Fc region subunits of the CyCAT hemimolecule according to this disclosure are capable of stably associating with each other.

[0634] In some embodiments, the Fc region of the CyCAT hemimolecule according to this disclosure is an IgG Fc region. In some embodiments, the Fc region is an IgG1 Fc region. In some embodiments, the Fc region is human-derived. In some embodiments, the Fc region is a human IgG1 Fc region.

[0635] Heterodimer Fc region

[0636] According to this disclosure, the two Fc region subunits of the CyCAT hemimolecule can be contained in two distinct polypeptide chains. To improve the yield and purity of the recombinant hemimolecule, it is advantageous to introduce one or more modifications into the Fc region that promote the association of the two distinct polypeptides forming the Fc region subunits. Therefore, in some embodiments, this disclosure provides heterodimeric CyCAT hemimolecules, each relying on the use of two different variant Fc region subunits that will self-assemble to form a heterodimeric molecule.

[0637] In embodiments, the Fc region of the CyCAT hemimolecule according to this disclosure includes one or more modifications that promote association between the first and second Fc region subunits and / or the third and fourth Fc region subunits. In embodiments, the first and second Fc region subunits and / or the third and fourth Fc region subunits of the CyCAT hemimolecule may include one or more modifications that respectively promote association between the first and second Fc region subunits and / or the third and fourth Fc region subunits.

[0638] In the embodiments, the first Fc subunit and the second Fc subunit and / or the third Fc subunit and the fourth Fc subunit contain one or more modifications that reduce homodimerization or reduce homodimer formation between two identical polypeptide chains containing the same Fc subunit.

[0639] In the embodiments, the first Fc region subunit and the second Fc region subunit and / or the third Fc region subunit and the fourth Fc region subunit contain different amino acid modifications, making the heterodimeric Fc region more stable than the homodimeric Fc region.

[0640] In embodiments, the first and second Fc subunits and / or the third and fourth Fc subunits contain different amino acid modifications that promote association between the first and second Fc subunits and / or the third and fourth Fc subunits. Modifications may be present in the first and / or second and / or third and / or fourth Fc subunits. In embodiments, such modifications are present in the first and second Fc subunits. In embodiments, such modifications are present in the third and fourth Fc subunits. In embodiments, such modifications occur in the CH3 domain of each Fc subunit. Modifications can be performed by altering the nucleic acid encoding the polypeptide (e.g., through site-specific mutagenesis or through peptide synthesis).

[0641] Typically, in heterodimerization methods known in the art, the CH3 domains of one polypeptide chain (e.g., the immunoglobulin heavy chain) and another polypeptide chain are engineered in a complementary manner, such that a polypeptide containing an engineered CH3 domain cannot homodimerize with another polypeptide chain of the same structure. This promotes heterodimerization between a polypeptide containing an engineered CH3 domain and another polypeptide containing a CH3 domain (engineered in a complementary manner). Several methods for CH3 modification to promote heterodimerization are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, and WO 2013 / 096291 (which are incorporated herein by reference).

[0642] One of these heterodimerization methods known in the art is the so-called "mortar and pestle" (KiH) technique, which is described in detail in, for example, WO 96 / 027011; Ridgway, JB et al., Protein Eng. 9 (1996) 617-621; Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681; US5,731,168; US 7,695,936; WO 98 / 050431; Carter, J Immunol Meth 248, 7-15 (2001) (which are incorporated herein by reference) and provides several examples. The "mortar and pestle" technique broadly involves: (1) mutating the CH3 domain in each Fc region subunit to promote heterodimerization; and (2) combining the mutated Fc region subunits under conditions that promote heterodimerization. "Pepper" or "protrusion" is typically generated by replacing a small amino acid in the parent antibody with a larger amino acid (e.g., T366Y or T366W); "mortar" or "cavity" is generated by replacing a larger residue in the parent antibody with a smaller amino acid numbered according to the EU index (e.g., Y407T, T366S, L368A and / or Y407V).

[0643] In the embodiments, the modification present in the Fc region of the CyCAT hemimolecule according to this disclosure is a "mortar and pestle" modification, which includes a "mortar mutation" in one of the two Fc region subunits and a "mortar mutation" in the other complementary Fc region subunit. Mortar and pestle modifications can be performed by altering the nucleic acid encoding the polypeptide (e.g., through site-specific mutagenesis or through peptide synthesis). In the embodiments, the CH3 domain of each Fc region subunit is modified according to the mortar and pestle technique.

[0644] In one embodiment, in the CH3 domain of the first Fc subunit and / or the third Fc subunit, the threonine residue at position 366 is replaced by a tryptophan residue (T366W), and in the CH3 domain of the second Fc subunit, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), as numbered according to the EU index. In another embodiment, in the CH3 domain of the second or fourth Fc subunit, the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A), as numbered according to the EU index. In the embodiments, in the CH3 domain of the first Fc subunit and / or the third Fc subunit, the serine residue at position 354 is replaced by a cysteine ​​residue (S354C), and in the CH3 domain of the second Fc subunit and / or the fourth Fc subunit, the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C), which are numbered according to the EU index. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two Fc subunits, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0645] In a more specific embodiment, this disclosure provides a CyCAT half-molecule wherein, in the CH3 domain of the first Fc subunit and / or the third Fc subunit, a threonine residue at position 366 is replaced by a tryptophan residue (T366W), and a serine residue at position 354 is replaced by a cysteine ​​residue (S354C); and in the CH3 domain of the second Fc subunit and / or the fourth Fc subunit, a tyrosine residue at position 407 is replaced by a valine residue (Y407V), a threonine residue at position 366 is replaced by a serine residue (T366S), a leucine residue at position 368 is replaced by an alanine residue (L368A), and a tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C), wherein the numbers are specified according to the EU index.

[0646] In embodiments, this disclosure provides a CyCAT half-molecule wherein the αCD3-VH or αCD3-VL domain and the Fc region subunit containing the acetamipel mutation are present on the same polypeptide chain. In alternative embodiments, this disclosure provides a CyCAT half-molecule wherein the αCD3-VH or αCD3-VL domain and the Fc region subunit containing the acetamipel mutation are present on the same polypeptide chain. In embodiments, the Fab heavy chain and the αCD3-VH or αCD3-VL domain and the Fc region subunit containing the acetamipel mutation are present on the same polypeptide chain.

[0647] In an alternative embodiment, the Fab heavy chain and the Fc region subunit containing the mortar-mutation exist on the same polypeptide chain, and the Fc region subunit containing the mortar-mutation and the αCD3-VH or αCD3-VL domain exist on a second, different polypeptide chain.

[0648] Fc receptor binding and / or effector function

[0649] For certain therapeutic situations, it may be necessary to reduce or inhibit the binding of the IgG Fc region to normal or wild-type Fc receptors and / or to complement components (e.g., C1q). For example, it may be necessary to reduce or prevent the binding of the Fc region to one or more Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa). In particular, when a pair of complementary hemimolecules co-binds to receptors of immune effector cells (e.g., TCRs) according to this disclosure, it is recommended to prevent FcγRIIIa binding to eliminate or significantly reduce ADCC activity and / or prevent C1q binding to eliminate or significantly reduce CDC activity. The reduced or eliminated effector function may include, but is not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced or eliminated antibody-dependent cell-mediated cytotoxicity (ADCC), and reduced or eliminated antibody-dependent phagocytosis (ADCP). In some embodiments, the reduced or eliminated effector function is one or more selected from the group consisting of CDC, ADCC, and ADCP. In one embodiment, the reduced or eliminated effector is ADCC. In another embodiment, the reduced or eliminated effector is CDC. In yet another embodiment, the reduced or eliminated effector is ADCP. In a further embodiment, the reduced or eliminated effectors are CDC, ADCC, and ADCP.

[0650] In embodiments, the Fc region of the half according to this disclosure is engineered to have a reduced binding affinity to the Fc receptor and / or C1q and / or reduced effector function compared to an unengineered Fc region. In embodiments, the Fc region of the half according to this disclosure is engineered to have reduced effector function compared to an unengineered Fc region. In embodiments, the Fc region of the half according to this disclosure contains one or more amino acid mutations that reduce the binding affinity of the Fc region to the Fc receptor and / or the Fc region to C1q, and / or reduce its effector function. Generally, the same one or more amino acid mutations are present in each of the two Fc region subunits forming the Fc region. In embodiments, one or more amino acid mutations reduce the binding affinity of the Fc region to the Fc receptor. In embodiments, the engineered Fc region substantially does not bind to the Fc receptor and / or C1q and / or induces effector function. In embodiments, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In an embodiment, the Fc receptor is an Fcγ receptor. In embodiments, the Fc receptor is an activated human Fcγ receptor, more particularly human FcγRIIIa, FcγRI, or FcγRIIa, and most particularly human FcγRIIIa. In embodiments, the binding affinity of the Fc region to complement components is reduced or eliminated, particularly the binding affinity of the Fc region to C1q. In embodiments, the reduced or eliminated effector function is one or more selected from the group consisting of reduced or eliminated CDC, reduced or eliminated ADCC, and reduced or eliminated ADCP. In specific embodiments, the reduced or eliminated effector functions are reduced ADCC, CDC, and ADCP. In embodiments, the Fc region of HB1 and / or HB2 according to this disclosure contains one or more amino acid mutations that reduce the binding affinity of the Fc region to the Fc receptor and / or the Fc region to C1q, and / or reduce effector function.

[0651] In embodiments, amino acid mutations are amino acid substitutions. In embodiments, the Fc region of the hemisphere according to this disclosure contains one or more amino acid mutations that reduce the binding affinity of the Fc region to the Fc receptor and / or the Fc region to C1q, and / or reduce its effector function, wherein each Fc region subunit contains an amino acid substitution at a position selected from the group consisting of 234, 235, 237, 330, and 331, numbered according to the EU index.

[0652] In embodiments, each Fc subunit of the hemisphere according to this disclosure comprises an amino acid substitution at a position selected from the group consisting of L234, L235, and G237 (numbered according to the EU index). In embodiments, each Fc subunit comprises amino acid substitutions L234A and L235E, wherein the substitutions are numbered according to the EU index. In embodiments, each Fc subunit comprises amino acid substitutions L234A, L235E, and G237A, wherein the substitutions are numbered according to the EU index. In embodiments, each Fc subunit comprises an amino acid substitution at a position selected from the group consisting of 330 and 331, wherein the substitutions are numbered according to the EU index. In embodiments, each Fc subunit comprises an amino acid substitution at positions 330 and 331, wherein the substitutions are numbered according to the EU index. In embodiments, the amino acid substitution is A330S or P331S. In embodiments, the Fc region of the hemisphere according to this disclosure comprises one or more amino acid mutations in each Fc region subunit, which reduce the binding affinity of the Fc region to the Fc receptor and / or the Fc region to C1q, and / or reduce effector function, wherein the one or more amino acid mutations are L234A, L235E, G237A, A330S, and P331S. In embodiments, the Fc region of the hemisphere according to this disclosure consists of one or more amino acid mutations in each Fc region subunit, which reduce the binding affinity of the Fc region to the Fc receptor and / or the Fc region to C1q, and / or reduce effector function, wherein the one or more amino acid mutations are L234A, L235E, G237A, A330S, and P331S. In embodiments, the Fc region is the IgG1 Fc region, particularly the human IgG1 Fc region. Mutant Fc regions or Fc region subunits can be prepared using genetic or chemical methods well known in the art through amino acid deletion, substitution, insertion, or modification. Genetic methods can include site-specific mutagenesis of coding DNA sequences, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, through sequencing.

[0653] In the embodiments, the Fc subunit according to this disclosure comprises the following amino acid sequence (Fc-AEASS-62): PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 62)

[0654] In the embodiments, the Fc subunit according to this disclosure comprises the following amino acid sequence (Fc-AEASS-PAC): PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 63)

[0655] In the embodiments, the Fc subunit according to this disclosure comprises the following amino acid sequence (Fc-AEASS): PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 64)

[0656] Functionality

[0657] According to this disclosure, a pair of complementary CyCAT hemimolecules can be used for the prevention and treatment of diseases mediated by biological pathways involving a target antigen. This can be preferably achieved by recruiting cytotoxic immune cells (e.g., T cells) to cells expressing the target antigen (preferably a tumor or cancer-associated antigen, either TAA or CAA, respectively).

[0658] The bioactivity of a pair of complementary CyCAT hemimolecules according to this disclosure can be measured by various assays known in the art, including those described in the examples of this application. Methods for determining functional activity may utilize conjugation assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA)), fluorescence activated cell sorting (FACS), and other methods well known in the art (see Hampton, R. et al. (1990; Serological Methods a Laboratory Manual, APS Press, St. Paul, Minnesota) and Maddox, DE et al. (1983; J. Exp. Med. 158:1211-1216). Alternatively, the assay may test the ability of a pair of complementary CyCAT hemimolecules disclosed herein to elicit a biological response in vivo or in vitro by binding to a set of biological target antigens. Biological activities may include, for example, inducing T cell proliferation, inducing signaling in T cells, inducing the expression of activation markers in T cells, inducing T cell secretion of cytokines, inhibiting signaling in target cells (e.g., tumor cells or tumor stromal cells), inhibiting target cell proliferation, inducing target cell lysis, and inducing tumor regression and / or improving survival.

[0659] In an embodiment, this disclosure provides a method for inducing the lysis of target cells (e.g., tumor cells), the method comprising contacting the cells with a pair of complementary CyCAT hemimolecules according to this disclosure in the presence of cytotoxic T cells.

[0660] In an embodiment, this disclosure provides a method for inhibiting the proliferation of target cells (e.g., tumor cells), the method comprising contacting the cells with a pair of complementary CyCAT hemimolecules according to this disclosure in the presence of cytotoxic T cells.

[0661] In an embodiment, this disclosure provides a method for inducing a cellular response in cytotoxic T cells, the method comprising contacting the cytotoxic T cells with a pair of complementary CyCAT hemimolecules according to this disclosure in the presence of target cells (e.g., tumor cells). In an embodiment, the cytotoxic T cells are cytotoxic CD8+ T cells.

[0662] In the embodiments, the cellular response is selected from the group consisting of: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers.

[0663] In an embodiment, this disclosure provides a method for inducing human T cell proliferation in the presence of target cells (e.g., tumor cells), the method comprising contacting the T cells with a pair of complementary CyCAT hemimolecules according to this disclosure in the presence of the T cells.

[0664] In an embodiment, this disclosure provides a method for stimulating a primary T cell response in the presence of a target cell (e.g., a tumor cell), the method comprising contacting the T cell with a pair of complementary CyCAT hemimolecules according to this disclosure in the presence of the T cell.

[0665] In an embodiment, this disclosure provides a method for redirecting the cytotoxic activity of T cells to target cells (e.g., tumor cells), the method comprising contacting the cancer cells with a pair of complementary CyCAT hemimolecules according to this disclosure in the presence of the T cells.

[0666] In embodiments, this disclosure provides the use of a pair of complementary CyCAT hemimolecules according to this disclosure for treating cancers in subjects that are positive for at least two tumor-associated antigens (TAAs), the use including:

[0667] (a) Selecting subjects with cancer,

[0668] (b) Collect one or more biological samples from the subject.

[0669] (c) Identify cancer cells expressing at least two tumor-associated antigens in one or more samples, and

[0670] (d) Administer to the subject an effective amount of a pair of CyCAT hemimolecules according to this disclosure.

[0671] In an embodiment, the cancer cells express a first TAA and a second TAA. In a preferred embodiment, the first TAA and the second TAA are different. In an embodiment, in a pair of complementary CyCAT hemimolecules according to this disclosure, HB1 binds to the first TAA and HB2 binds to the second TAA.

[0672] Fusion Molecules

[0673] The hemimolecules according to this disclosure can be fused with one or more additional moieties. Such fusion proteins can be prepared in any suitable manner, including genetic or chemical methods. The linked moieties may contain secretory or leader sequences, sequences that facilitate detection, expression, isolation, or purification, or sequences that confer, for example, increased protein stability during recombination. Non-limiting examples of potential moieties include β-galactosidase, glutathione S-transferase, luciferase, T7 polymerase fragments, secretory signal peptides, toxins, antibodies or antibody fragments, reporter enzymes, moieties capable of binding metal ions (such as polyhistidine tags), tags suitable for detection and / or purification, moieties that increase protein solubility, or moieties containing enzymatic cleavage sites. It should be understood that such additional moieties may or may not provide additional functionality to the hemimolecules according to this disclosure, and may or may not modify the properties of HB1 and / or HB2. The hemimolecules and moieties according to this disclosure can be fused via peptide linkers as described herein.

[0674] produce

[0675] The methods for generating the CyCAT hemimolecules disclosed herein are well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). The CyCAT hemimolecules according to this disclosure can be obtained, for example, by solid-phase peptide synthesis or recombinant generation. For recombinant generation, one or more nucleic acid sequences encoding the CyCAT hemimolecule are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Complementary CyCAT hemimolecules according to this disclosure are preferably generated individually.

[0676] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the CyCAT hemisome molecule and appropriate transcription / translation control signals. Such methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / gene recombination. See, for example, these techniques described in Maniatis et al., *Molecular Cloning: A Lab Manual*, Cold Spring Harbor Laboratory, New York (1989); and Ausubel et al., *Current Protocols in Molecular Biology*, Greene Publishing Associates and Wiley Interscience, New York (1989). Vectors can be introduced into suitable host cells (e.g., prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cells) using methods well known in the art (see, for example, "Current Protocol in Molecular Biology", Ausubel et al. (eds.), Greene Publishing Assoc. and John Wiley Interscience, New York, 1989 and 1992). Many cloning vectors are known to those skilled in the art, and appropriate cloning vectors are selected as needed. The coding sequence can be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operon, such that a DNA sequence encoding the desired protein or polypeptide is transcribed into RNA in a host cell transformed with one or more vectors containing the expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. Depending on the chosen expression system and host cell, the hemimolecule according to this disclosure is produced by growing a host cell transformed with the aforementioned expression vector under conditions for expressing the target hemimolecule. The hemimolecule is then isolated from and purified from the host cell. If the expression system secretes the hemimolecule into the growth medium, the protein can be purified directly from the medium. If the CyCAT hemimolecule is not secreted, it is separated from cell lysates or recovered from cell membrane fractions. The selection of appropriate growth conditions and recovery methods is within the scope of the art. It should be noted that the CyCAT hemimolecule according to this disclosure is not a naturally occurring protein. Typically, the hemimolecule according to this disclosure is a recombinant protein, a synthetic protein, or a semi-synthetic protein.In an embodiment, a method for generating a hemimolecule according to the present disclosure is provided, wherein the method includes culturing a host cell comprising a vector composition comprising one or more vectors encoding one or more nucleic acid sequences encoding a CyCAT hemimolecule according to the present disclosure under conditions suitable for expressing a CyCAT hemimolecule, and recovering the CyCAT hemimolecule from the host cell or host cell culture medium. In an embodiment, the method for generating a CyCAT hemimolecule according to the present disclosure further includes the step of isolating the generated CyCAT hemimolecule from the host cell or culture medium. The recovered hemimolecule, as described herein, can be purified using purification techniques known in the art, such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The conditions used to purify a particular hemimolecule will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens that bind to the hemimolecule can be used. For example, for affinity chromatography purification of a CyCAT hemimolecule containing an IgG Fc region, a matrix having protein A or protein G can be used. The purity of CyCAT half molecules can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high-performance liquid chromatography, etc.

[0677] Treatment

[0678] A pair of complementary CyCAT hemimolecules according to this disclosure can be used in treatment methods. In embodiments, this disclosure provides a pair of CyCAT hemimolecules according to this disclosure for treating a disease. In embodiments, this disclosure provides a pair of complementary CyCAT hemimolecules for use in treating a disease in a subject of need. A pair of CyCAT hemimolecules according to this disclosure can be used to treat cancer. In embodiments, this disclosure provides the use of a pair of CyCAT hemimolecules for manufacturing a medicament. In embodiments, this disclosure provides a pair of CyCAT hemimolecules for use as a medicament. In embodiments, this disclosure provides a pair of CyCAT hemimolecules for use as a medicament for treating a disease in a subject of need. In embodiments, the disease is associated with an unwanted antigen. In a preferred embodiment, the disease is associated with two unwanted antigens.

[0679] In this embodiment, the disease to be treated is a proliferative disease. In a specific embodiment, the disease is cancer or a tumor. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, leukemia, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.

[0680] In an embodiment, this disclosure provides a pair of complementary hemimolecules according to this disclosure for use in a method of treating a subject with a disease, the method comprising administering a therapeutically effective amount of the pair of complementary CyCAT hemimolecules to the subject.

[0681] In embodiments, the method further includes administering a therapeutically effective amount of at least one additional therapeutic agent to the subject. The subject requiring treatment is typically a mammal, and more particularly a human. For use in the treatment, a pair of complementary hemimolecules according to this disclosure will be formulated, administered, and applied in accordance with good medical practice. In embodiments, of the pair of complementary hemimolecules according to this disclosure, two complementary hemimolecules are administered individually. In embodiments, two complementary hemimolecules are administered sequentially. In embodiments, two complementary hemimolecules are administered one after the other.

[0682] In embodiments, this disclosure provides a method for inducing tumor regression in a patient with cancer, the method comprising administering a therapeutically effective amount of a pair of CyCAT hemimolecules to the subject. In embodiments, this disclosure provides a method for improving the survival of a subject with cancer, the method comprising administering a therapeutically effective amount of a pair of complementary hemimolecules to the subject. In embodiments, this disclosure provides a method for initiating, stimulating, or inducing an immune response in a subject with cancer, the method comprising administering a therapeutically effective amount of a pair of complementary hemimolecules according to this disclosure to the subject. In embodiments, this disclosure provides a method for enhancing or inducing anti-cancer immunity in a subject with cancer, the method comprising administering a therapeutically effective amount of a pair of complementary hemimolecules according to this disclosure to the subject.

[0683] In an embodiment, this disclosure provides a method for treating cancer in a subject caused by cancer cells expressing a first antigen and a second antigen on their cell surface, wherein a first half-molecule and a second half-molecule are administered simultaneously or sequentially to a subject having cancer cells expressing the first antigen and the second antigen on their cell surface. The first half-molecule comprises a protective domain, αCD3-VH or αCD3-VL, and a Fab that binds to the first antigen; the second half-molecule comprises a corresponding complementary αCD3-VH or αCD3-VL, and a Fab that binds to the second antigen. In an embodiment, the first half-molecule and the second half-molecule are not covalently linked. In an embodiment, the first half-molecule and the second half-molecule are not covalently linked before and after administration, but instead form a heterodimer on the surface of the cancer cells.

[0684] Pharmaceutical Composition

[0685] In embodiments, this disclosure provides a pharmaceutical composition comprising a hemimolecule according to this disclosure and at least one pharmaceutically acceptable carrier. In embodiments, this disclosure provides a pharmaceutical composition comprising HB1 and HB2 according to this disclosure, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In other embodiments, HB1 and HB2 may be formulated as separate pharmaceutical compositions or contained in separate pharmaceutical compositions.

[0686] In embodiments, this disclosure provides a first pharmaceutical composition comprising HB1 according to this disclosure and at least one pharmaceutically acceptable carrier. In embodiments, this disclosure provides a second pharmaceutical composition comprising HB2 according to this disclosure and at least one pharmaceutically acceptable carrier. The pharmaceutical compositions may further comprise at least one other pharmaceutically active compound according to this disclosure. The pharmaceutical compositions according to this disclosure can be used for the diagnosis, prevention, and / or treatment of diseases associated with the presence of AG1 and / or AG2.

[0687] In particular, this disclosure provides (a) a first pharmaceutical composition comprising HB1 according to this disclosure and a second pharmaceutical composition comprising HB2 according to this disclosure, suitable for preventive, therapeutic and / or diagnostic use in mammals (more particularly humans).

[0688] In the embodiments, this disclosure provides a first pharmaceutical composition comprising HB1 according to this disclosure and a second pharmaceutical composition comprising HB2 according to this disclosure for use in the prevention and / or treatment of diseases associated with the presence of undesirable AG1 and AG2.

[0689] In embodiments, this disclosure provides a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2 for use as a medicine. In embodiments, this disclosure provides a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2 for use in the prevention and / or treatment of a disease. In embodiments, this disclosure provides a method for treating a disease in a subject of need, the method using a pharmaceutical composition comprising HB1 and a pharmaceutical composition comprising HB2.

[0690] In some embodiments, the disease is an autoimmune disease, an inflammatory disease, cancer, a vascular disease, an infectious disease, thrombosis, myocardial infarction, and / or diabetes. In some embodiments, the disease is a proliferative disease. In certain embodiments, the disease is cancer or a tumor. In some embodiments, the disease is cancer. In some embodiments, the cancer expresses AG1. In some embodiments, the cancer expresses AG2. In some embodiments, the cancer expresses both AG1 and AG2.

[0691] The pharmaceutical compositions according to this disclosure may comprise therapeutically effective amounts of HB1 and HB2 (dissolved in a pharmaceutically acceptable carrier). In embodiments, this disclosure provides a kit comprising HB1 and HB2 according to this disclosure. In embodiments, this disclosure provides a kit comprising a first pharmaceutical composition containing HB1 and a second pharmaceutical composition containing HB2. In embodiments, this disclosure provides a method for treating autoimmune diseases, inflammatory diseases, cancer, vascular diseases, infectious diseases, thrombosis, myocardial infarction, and / or diabetes in a subject of need, the method using a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2.

[0692] Dosage

[0693] To prevent or treat disease, the appropriate dosage of HB1 and HB2 as disclosed herein needs to be an effective amount to ensure the intended purpose, and will depend on the type of disease to be treated, the route of administration, the subject's weight, the severity and duration of the disease, whether the administration of HB1 and HB2 is for preventive or therapeutic purposes, prior or concurrent therapeutic interventions, the subject's clinical history and response to the pair of complementary hemimeric molecules, and the judgment of the attending physician.

[0694] Effective doses and schedules for administering the pharmaceutical compositions comprising HB1 and HB2 disclosed herein can be determined empirically; for example, patient progression can be monitored through periodic assessments, and doses adjusted accordingly. Furthermore, interspecies scaling of doses can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. [Pharmaceutical Research] 8:1351). In embodiments, this disclosure provides HB1 and HB2 according to this disclosure, wherein said HB1 and HB2 are administered at doses sufficient to achieve therapeutically effective serum levels. Administration of HB1 and HB2 according to this disclosure encompasses single administration, in which case administration of HB1 occurs before, simultaneously with, and / or after administration of HB2, or vice versa. In some embodiments, HB1 and HB2 are administered intravenously. In some embodiments, HB1 and HB2 are administered subcutaneously.

[0695] Combination therapy

[0696] HB1 and HB2 according to this disclosure can be administered in combination with one or more other therapeutic agents. "Therapeutic agent" encompasses any agent administered to treat symptoms or diseases in a subject requiring such treatment. In some embodiments, the additional therapeutic agent is an immunomodulator, cell inhibitor, cell adhesion inhibitor, cytotoxic agent, apoptosis activator, or agent that increases the sensitivity of cells to apoptosis-inducing agents. Such additional therapeutic agents are suitably present in combination in an amount effective for the intended purpose. Combination therapy encompasses combined administration (where two or more therapeutic agents are contained in the same or separate compositions) and single administration (in which case, administration of HB1 and HB2 according to this disclosure may occur before, simultaneously with, and / or after administration of additional therapeutic agents). HB1 and HB2 according to this disclosure can also be used in combination with radiotherapy.

[0697] diagnosis

[0698] In embodiments, this disclosure provides the use of HB1 and HB2 according to this disclosure for diagnosing diseases. In embodiments, this disclosure provides the use of HB1 and HB2 for detecting AG1 and / or AG2. In embodiments, this disclosure provides a method for detecting AG1 and / or AG2 in a subject or sample, the method comprising the step of contacting the subject or sample with a pair of complementary hemimolecules according to this disclosure. In embodiments, this disclosure provides a method for diagnosing a disease in a subject, the method comprising the step of contacting the subject or sample with a pair of complementary hemimolecules according to this disclosure.

[0699] Work examples

[0700] The following are examples of molecules and methods based on this disclosure. It should be understood that various other embodiments may be practiced given the general description provided herein. DNA is manipulated using standard methods as described below: Sambrook et al., *Molecular Cloning: A Laboratory Manual*; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. General information regarding the nucleotide sequences of the light and heavy chains of human immunoglobulins is given below: Kabat, E.A. et al., (1991) *Sequences of Proteins of Immunological Interest*, 5th Edition, NIH Publication No. 91-3242.

[0701] Combined structural domains:

[0702] For human HER2 (UniPROT: P04626) binding, the coding trastuzumab (HERCEPTIN) was used. ® The nucleotide sequences of the VH and VL domains of trastuzumab are described in Baselga et al., 1998, Cancer Res [Cancer Research] 58(13): 2825-2831. Trastuzumab and its preparation method are disclosed in US Patent 5,821,337.

[0703] For human EGFR (UniPROT: P00533) binding, the encoding of cetuximab (Erbitux) was used. ® The nucleotide sequences of the VH and VL domains of cetuximab are described in US Patent 7,060,808.

[0704] For chicken egg lysozyme binding, nucleotide sequences encoding the VH and VL domains from the internal control antibody MOR03207 were used.

[0705] For CD3 binding, nucleotide sequences encoding the VH and VL domains of human anti-CD3 antibodies according to Tables 2 and 3 of this application are used. Anti-CD3 antibodies and methods for their preparation are described in WO 2022 / 063819.

[0706] An overview of the amino acid sequence of the CyCAT half-molecule prepared according to the examples described herein is listed in the following sections.

[0707] Construction of CyCAT half-molecules

[0708] CyCAT half-body form lacking protected structural domains

[0709] Various CyCAT half-molecules containing Fab fragments and unpaired αCD3 antibody variable domains (“αCD3-SVD”) (e.g., αCD3-VH or αCD3-VL domains) were designed and generated.

[0710] B027 format

[0711] The structure of the CyCAT half-molecule in the form of B027 is provided in Figure 2 The half-molecule is composed of a Fab fragment as the targeting portion, which connects to a single αCD3-VL domain at the C-terminus of its Fab heavy chain via a short peptide linker. Figure 2 A) or a single αCD3-VH domain ( Figure 2The N-terminus of A') is fused. Each hemimolecule consists of two polypeptide chains: a main chain comprising the Fab heavy chain and αCD3-SVD; and a second polypeptide comprising the Fab light chain of trastuzumab or cetuximab (whichever is more specific). Exemplary sequences of the CyCAT hemimolecule in the form of B027 used in examples of the present invention are summarized below.

[0712] Half-body 1 (HB-1): (Fab) αHER2 / αCD3 VL低 )

[0713] Main chain: VH （曲妥珠单抗） – CH1 – Connector （GQPSG（SEQ ID NO: 45）） – VL （αCD3低）

[0714] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 65)

[0715] Half-body 2 (HB-2): (Fab αHER2 / αCD3 VH低 )

[0716] Main chain: VH （曲妥珠单抗） – CH1 – Connector （GQPSG（SEQ ID NO: 45）） – VH （αCD3低）

[0717] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFTFKSYYMSWVRQAPGKGLEWVANIDYQSQHAYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 66)

[0718] Half - body 3 (HB - 3): (Fab αEGFR / CD3 VL低 )

[0719] Backbone: VH （西妥昔单抗） – CH1 – linker （GQPSG（SEQ ID NO: 45）） – VL （αCD3低）

[0720] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 67)

[0721] Half - body 4 (HB - 4): (Fab αEGFR / αCD3 VH低 )

[0722] Main chain: VH （西妥昔单抗） – CH1 – linker （GQPSG（SEQ ID NO: 45）） – VH （αCD3低）

[0723] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFTFKSYYMSWVRQAPGKGLEWVANIDYQSQHAYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 68)

[0724] Fab light chain of trastuzumab

[0725] Light chain: VL （曲妥珠单抗） – CL

[0726] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 69)

[0727] Fab light chain of cetuximab

[0728] Light chain: VL （西妥昔单抗） – CL

[0729] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 70)

[0730] Form B036

[0731] The structure of the CyCAT half-molecule in the form of B036 is provided in Figure 2 A and Figure 2 In A', this half-form is incorporated into the complete human IgG1 Fc region. The Fab fragment is coupled with a single unpaired αCD3-VL domain ( Figure 2 A) or αCD3-VH domain ( Figure 2 The fusion of A') is achieved via a first peptide linker. The unpaired αCD3-SVD then fuses with the Fc subunit carrying the acetamipel mutation via a second peptide linker (containing a short IgG hinge sequence). The second Fc subunit carrying the acetamipel mutation also contains a short IgG hinge linker sequence at its N-terminus. The use of cysteine ​​residues in the linker sequence allows for further stabilization of the Fc region via the formation of interchain disulfide bonds. Exemplary sequences of the B036 form of hemimolecules used in the examples disclosed herein are summarized below. Each hemimolecule consists of three polypeptide chains: a first backbone containing a Fab heavy chain, αCD3-SVD, and a first Fc subunit; a second backbone containing a second Fc subunit; and a third chain containing a Fab light chain of trastuzumab, cetuximab, or α-lysozyme (as the case may be).

[0732] Half-body 5 (HB-5): (Fab αHER2 / αCD3 VL高 )

[0733] Main chain 1: VH （曲妥珠单抗） – CH1 – Connector (G4S)4（SEQ ID NO: 46） – VL （αCD3VL高） -Connector （PAPDA-铰链区截短） –CH2 （AEASS） –CH3 （杵）

[0734] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71)

[0735] Half body 6 (HB-6): (Fab αEGFR / αCD3 VH高 )

[0736] Backbone 1: VH （西妥昔单抗） –CH1–Linker (G4S)4（SEQ ID NO: 46） –VH<0000l62> –Linker （PAPDA-铰链区截短） –CH2 （AEASS） –CH3 （杵） ]>

[0737] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 72)

[0738] Half body 7 (HB-7): (Fab αEGFR / CD3 VL高 )

[0739] Backbone 1: VH （西妥昔单抗） –CH1–Linker (G4S)4（SEQ ID NO: 46） –VL （αCD3高） [[ID=1又6]]–Linker （PAPDA-铰链区截短） –CH2 （AEASS） –CH3 （杵）

[0740] It should be noted that there may be some inaccuracies in the translation due to the complexity and specific context of the patent text. It is recommended to review and verify with the relevant technical experts for more accurate understanding.QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 73)

[0741] Half body 8 (HB-8): B036 (α-lysozyme / αCD3 VL高 )

[0742] Backbone 1: VH （溶菌酶） – CH1 – linker (G4S)4（SEQ ID NO: 46） – VL （αCD3高） – linker （PAPDA-铰链区截短） –CH2 （AEASS） – CH3 （杵）

[0743] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74)

[0744] Backbone 2: Linker （铰链区截短） – CH2 （AEASS） – CH3 （臼）

[0745] KTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 75) ​Fab light chain of trastuzumab

[0747] Light chain: VL （曲妥珠单抗） – CL

[0748] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 69)

[0749] Fab light chain of cetuximab

[0750] Light chain: VL （西妥昔单抗） – CL

[0751] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 70)

[0752] Fab light chain of α-lysozyme

[0753] Light chain: VL （溶菌酶） – CL

[0754] DIELTQPPSVSVAPGQTARISCSGDNLPAYTVTWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCASWDPSSGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 76)

[0755] Form B039

[0756] The structure of the CyCAT half-molecule in the form of B039 is provided in Figure 2 C and Figure 2 In C', this hemisphere is composed of two identical Fab fragments as the target portion and two identical adjacent αCD3-SVDs (e.g., two identical αCD3-VH domains). Figure 2 C') or two identical αCD3-VL domains ( Figure 2 C)) Composition. The αCD3-SVD is located at the center between the two Fab arms and the Fc region. The fusion of each of the two Fab fragments with either of the two identical αCD3-SVDs is achieved via a peptide linker. Each of the two identical αCD3-SVDs is sequentially fused with one of the two Fc region subunits using two additional peptide linkers (containing a short IgG hinge sequence). Based on its symmetrical structure, this form has no clubbing mutation in its Fc region. The exemplary sequence of the B039 form of the hemimolecule used in the examples of this disclosure is summarized below. Each hemimolecule consists of four polypeptide chains: two identical backbones, each containing a Fab heavy chain, an αCD3-SVD, and a first Fc region subunit; and two identical polypeptide chains containing the Fab light chain of cetuximab.

[0757] Half-body 9 (HB-9): B039 (Fab αEGFR / αCD3 VH高 )

[0758] Main chain: VH （西妥昔单抗） – CH1 – Connector (G4S)4（SEQ ID NO: 46） – VH （αCD3高） -Connector （PAPDA-铰链区截短） –CH2 （AEASS） – CH3

[0759] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 77)

[0760] Half body 10 (HB-10): B039 (Fab αEGFR / αCD3 VL高 )

[0761] Main chain: VH （西妥昔单抗） –CH1–Linker (G4S)4（SEQ ID NO: 46） –VL （αCD3高） –Linker （PAPDA-铰链区截短） –CH2 （AEASS） –CH3

[0762] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 78)

[0763] Fab light chain of cetuximab

[0764] Light chain: VL （西妥昔单抗） – CL

[0765] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 70)

[0766] Form B063

[0767] The structure of the CyCAT half-molecule in the form of B063 is provided in Figure 2 B and Figure 2 In B', this hemisphere consists of two identical Fab fragments as the targeting portion and a single αCD3-SVD (e.g., an αCD3-VH) located between one of the two Fab fragments and an Fc region subunit. Figure 2 B') or an αCD3-VL ( Figure 2 B)) Composition. The fusion of the second Fab fragment with the Fc subunit carrying the mortar mutation is achieved via a short hinge-derived sequence. αCD3-SVD then fuses with the Fc subunit carrying the mortar mutation. Exemplary sequences of the CyCAT hemimolecule in the form of B063 used in the examples disclosed herein are summarized below. Each hemimolecule consists of four polypeptide chains: a first backbone comprising a first Fab heavy chain, a single αCD3-SVD, and a first Fc subunit; a second backbone comprising a second Fab heavy chain and a second Fc subunit; and two identical polypeptides, each comprising a Fab light chain of cetuximab.

[0768] Half-body 11 (HB-11): B063 (Fab αEGFR / αCD3 VH高 )

[0769] Main chain 1: VH （西妥昔单抗） – CH1 – Connector (G4S)4（SEQ ID NO: 46） – VH （αCD3高） -Connector （PAPDA-铰链区截短） –CH2 （AEASS） – CH3 （杵）

[0770] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 72)

[0771] Half body 12 (HB-12): B063 (Fab αEGFR / αCD3 VL高 )

[0772] Backbone 1: VH （西妥昔单抗） –CH1–Linker (G4S)4（SEQ ID NO: 46） –VL （αCD3高） –Linker （PAPDA-铰链区截短） –CH2 （AEASS） –CH3 （杵）

[0773] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 73)

[0774] The second backbone of the semi-body molecule in the B063 format

[0775] Backbone 2: VH （西妥昔单抗） – CH1 – linker （铰链区截短） – CH2 （AEASS） – CH3 （臼）

[0776] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPEEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 79)

[0777] cetuximab Fab light chain

[0778] Light chain: VL （西妥昔单抗） – CL

[0779] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 70)

[0780] B038 format

[0781] The structure of the CyCAT half-molecule in the form of B038 is provided in Figure 2 D and Figure 2 In D'. This hemisome consists of the following: a human IgG1 Fc region located in a Fab arm and a single unpaired αCD3-VL ( Figure 2 D) or αCD3-VH ( Figure 2The center between the D') domains. The fusion between the C-terminus of the Fab heavy chain and the N-terminus of one of the Fc region subunits carrying the mortar mutation is achieved using a short peptide linker containing a hinge-derived amino acid sequence. The second Fc region subunit containing the mortar mutation also contains a short hinge-derived peptide linker at its N-terminus. Exemplary sequences of CyCAT hemimolecules in the form of B038 used in the examples of this disclosure are summarized below. Each hemimolecule consists of three polypeptide chains: a first backbone containing a Fab heavy chain of trastuzumab or α-lysozyme, a first Fc region subunit, and a single αCD3-SVD; a second backbone containing a second Fc region subunit; and a third polypeptide containing a Fab light chain of trastuzumab or α-lysozyme (as applicable).

[0782] Half-body 13 (HB-13): B038 (Fab αHER2 / αCD3 VL高 )

[0783] Main chain: VH （曲妥珠单抗） – CH1 – Connector （铰链） – CH2 （AEASS） – CH3 （杵） -Connector （(G4S)4）（SEQ ID NO: 46） – VL （αCD3高）

[0784] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 80)

[0785] Half-body 14 (HB-14): B038 (Fab αHER2 / αCD3 VL低 )

[0786] Backbone: VH （曲妥珠单抗） –CH1–Linker （铰链） –CH2 （AEASS） –CH3 （杵） –Linker (G4S)4（SEQ ID NO: 46） –VL （αCD3低）

[0787] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 81)

[0788] Half body 15 (HB-15): B038 (Fab αHER2 / αCD3 VH高 )

[0789] Main chain: VH （曲妥珠单抗） – CH1 – Linker （铰链） – CH2 （AEASS） – CH3 （杵） – Linker （(G4S)4）（SEQ ID NO: 46） – VH （αCD3高）

[0790] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 82)

[0791] Half body 16 (HB-16): B038 (α-lysozyme / αCD3 VL高 )

[0792] Backbone: VH （溶菌酶） – CH1 – Linker （铰链） – CH2 （AEASS） – CH3 （杵） – Linker （(G4S)4）（SEQ ID NO: 46） –VL （αCD3高）

[0793] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO:83)

[0794] Half-body 17 (HB-17): B038 (α-lysozyme / αCD3 VL低 )

[0795] Backbone: VH （溶菌酶） – CH1 – Linker （铰链） – CH2 （AEASS） – CH3 （杵） – Linker （(G4S)4）（SEQ ID NO: 46） –VL （αCD3Vl低）

[0796] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO:84)

[0797] Half body 18 (HB-18): B038 (α-lysozyme / αCD3 VH高 )

[0798] Backbone: VH （溶菌酶） – CH1 – linker （铰链） – CH2 （AEASS） – CH3 （杵） – linker （(G4S)4）（SEQ ID NO: 46） –VH （αCD3VH高）

[0799] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 85)

[0800] The second backbone of the semi-body molecule in the B038 format

[0801] Second backbone: linker （铰链区截短） – CH2 （AEASS） – CH3 （臼）

[0802] KTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 75)

[0803] Fab light chain of α-lysozyme

[0804] Light chain: VL （溶菌酶） – CL

[0805] DIELTQPPSVSVAPGQTARISCSGDNLPAYTVTWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCASWDPSSGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 76)

[0806] Fab light chain of trastuzumab

[0807] Light chain: VL （曲妥珠单抗） – CL

[0808] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 69)

[0809] Form B064

[0810] The structure of the CyCAT moiety molecule of B064 is provided inFigure 2 E and Figure 2 In E'. As an alternative embodiment of the B038 form, this hemisome consists of two identical Fab fragments that allow for bivalent targeting. Exemplary sequences of the B064 form of hemisome molecules used in the examples of this disclosure are summarized below. Each hemisome molecule consists of four polypeptide chains: a first backbone comprising a first Fab heavy chain of trastuzumab or α-lysozyme, a first Fc region subunit containing a mordant mutation, and a single αCD3-SVD; a second backbone comprising a second Fab heavy chain of trastuzumab or α-lysozyme, and a second Fc region subunit containing a mordant mutation; and two identical polypeptides, each comprising a Fab light chain of trastuzumab or α-lysozyme (as applicable).

[0811] Half-body 19 (HB-19): B064 (Fab αHER2 / αCD3 VL高 )

[0812] Main chain 1: VH （曲妥珠单抗） – CH1 – Connector （铰链） – CH2 （AEASS） – CH3 （杵） - connector (G4S)4（SEQ ID NO: 46） -VL （αCD3高）

[0813] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 86)

[0814] Half-body 20 (HB-20): B064 (Fab αHER2 / αCD3 VL低）

[0815] Backbone 1: VH （曲妥珠单抗） – CH1 – Linker （铰链） – CH2 （AEASS） – CH3 （杵） - Linker (G4S)4（SEQ ID NO: 46） - VL （αCD3低）

[0816] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 87)

[0817] Half body 21 (HB-21): B064 (Fab αHER2 / αCD3 VH高 )

[0818] Backbone 1: VH （曲妥珠单抗） – CH1 – linker （铰链） – CH2 （AEASS） – CH3 （杵） - Linker (G4S)4（SEQ ID NO: 46） - VH （αCD3高）

[0819] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 88)

[0820] Half body 22 (HB-22) (B064) (α-lysozyme / αCD3 VL高 )

[0821] Backbone 1: VH （溶菌酶） – CH1 – Linker （铰链） – CH2 （AEASS） – CH3 （杵） – Linker (G4S)4（SEQ ID NO: 46） -VL （αCD3高）

[0822] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO:89)

[0823] Half body 23 (HB-23) (B064) (α-lysozyme / αCD3 VL低 )

[0824] Backbone 1: VH （溶菌酶） – CH1 – Linker （铰链） – CH2 （AEASS） – CH3 （杵） - Linker (G4S)4（SEQ ID NO: 46） -VL （αCD3低）

[0825] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO:90)

[0826] Half body 24 (HB-24) (B064) (α-lysozyme / αCD3 VH高 )

[0827] Backbone 1: VH （溶菌酶） – CH1 – Linker （铰链） – CH2 （AEASS） – CH3 （杵） – Linker (G4S)4（SEQ ID NO: 46） -VH （αCD3高）

[0828] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 91)

[0829] The second backbone of the semi - body molecule in B064 form

[0830] Backbone 2: VH （曲妥珠单抗） – CH1 – linker （铰链） – CH2 （AEASS） – CH3 （臼）

[0831] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)

[0832] Backbone 2: VH （溶菌酶） – CH1 – Linker （铰链） – CH2 （AEASS） – CH3 （臼）

[0833] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93)

[0834] Fab light chain of α-lysozyme

[0835] Light chain: VL （溶菌酶） – CL

[0836] DIELTQPPSVSVAPGQTARISCSGDNLPAYTVTWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCASWDPSSGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 76)

[0837] Fab light chain of trastuzumab:

[0838] Light chain: VL （曲妥珠单抗） – CL

[0839] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 69)

[0840] B050 format

[0841] The structure of the CyCAT half-molecule of B050 provides... Figure 2 F and Figure 2 In F'. As an alternative embodiment of the B064 form, the half consists of two identical adjacent αCD3-SVDs (e.g., two identical αCD3-VH domains) present at the C-terminus of the half. Figure 2 F') or two identical αCD3-VL domains ( Figure 2 The F) form is composed of two identical backbones. Due to its symmetrical structure and the presence of two identical backbones, this form does not have a club-and-groove mutation in its Fc region. Exemplary sequences of the CyCAT hemimolecule in the B050 form used in the examples disclosed herein are summarized below. Each hemimolecule consists of four polypeptide chains: two identical backbones containing the Fab heavy chain of trastuzumab or α-lysozyme, the Fc region subunit, and αCD3-SVD; and two identical polypeptides, each containing the Fab light chain of trastuzumab or α-lysozyme (whichever is more specific).

[0842] Half-body 25 (HB-25): B050 (Fab) αHER2 / αCD3 VL高 )

[0843] Main chain: VH （曲妥珠单抗） – CH1 – Connector （铰链） – CH2 （AEASS） – CH3 – Connector (G4S)4（SEQ ID NO: 46） -VL （αCD3高）

[0844] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 94)

[0845] Half body 26 (HB-26): B050 (Fab αHER2 / αCD3 VL低 )

[0846] Backbone: VH （曲妥珠单抗） – CH1 – linker （铰链） – CH2 （AEASS） – CH3 – linker (G4S)4（SEQ ID NO: 46） –VL （αCD3低）

[0847] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 95)

[0848] Half body 27 (HB-27): B050 (Fab αHER2 / αCD3 VH高 )

[0849] Main chain: VH （曲妥珠单抗） – CH1 – Linker （铰链） – CH2 （AEASS） – CH3 – Linker (G4S)4（SEQ ID NO: 46） –VH （αCD3高）

[0850] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO:96)

[0851] Half body 28 (HB-28): B050 (α-lysozyme / αCD3 VL高 )

[0852] Backbone: VH （溶菌酶） – CH1 – linker （铰链） – CH2 （AEASS） – CH3 – linker (G4S)4（SEQ ID NO: 46） -VL （αCD3高）

[0853] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO:97)

[0854] Half body 29 (HB-29): B050 (α-lysozyme / αCD3 VL低 )

[0855] Backbone: VH （溶菌酶） – CH1 – linker （铰链） – CH2 （AEASS） – CH3 – linker (G4S)4（SEQ ID NO: 46） –VL （αCD3低）

[0856] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO:98)

[0857] Half body 30 (HB-30): B050 (α-lysozyme / αCD3 VH高 )

[0858] Backbone: VH （溶菌酶） – CH1 – linker （铰链） – CH2 （AEASS） – CH3 – linker (G4S)4（SEQ ID NO: 46） –VH （αCD3高）

[0859] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 99)

[0860] Fab light chain of α-lysozyme

[0861] Light chain: VL （溶菌酶） – CL

[0862] DIELTQPPSVSVAPGQTARISCSGDNLPAYTVTWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCASWDPSSGVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ IDNO: 76)

[0863] Fab light chain of trastuzumab

[0864] Light chain: VL （曲妥珠单抗） – CL

[0865] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 69)

[0866] CyCAT half-body form (HB) containing protected structural domains GD )

[0867] The protective domain (GD) attached to the CyCAT hemibody as described herein spatially blocks the binding of unpaired αCD3-SVD present on the same hemibody to complementary αCD3-SVD present on the second hemibody molecule in the absence of target cells expressing both target antigens or expressing only one of the two target antigens. Preferably, the GD disclosed herein cannot bind to or interact with unpaired αCD3-SVD present on the same hemibody molecule to be shielded.

[0868] In examples of the present invention, this is illustrated by using human serum albumin (HSA) (SEQ ID NO: 17), HSA domain III (SEQ ID NO: 18), an inactive VL protective domain (e.g., having the amino acid sequence of SEQ ID NO: 14), an inactive VH protective domain (e.g., having the amino acid sequence of SEQ ID NO: 15), or an inactive VH protective domain (e.g., having the amino acid sequence of SEQ ID NO: 16).

[0869] Negative control: Protective domain half-body lacking αCD3-SVD

[0870] The amino acid sequences of the B027-based hemispheres described above contain VL protecting domains (GD... VL ) or VH protected structural domain (GD VH ) or VH pestle protective structural domain (GD VH-杵 ) Fab fragments that are specific to HER2 or EGFR fused together.

[0871] Each half-molecule consists of two polypeptide chains: a main chain containing the Fab heavy chain and protective domain of trastuzumab or cetuximab; and a second polypeptide containing the Fab light chain of trastuzumab or cetuximab (whichever is more specific).

[0872] GD-Half-body 1 (HB) GD -1): B027 (Fab) αHER2 / GD VL )

[0873] Main chain: VH （曲妥珠单抗） – CH1 – Connector （GQPSG（SEQ ID NO: 45）） – GD VL

[0874] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQ (SEQ ID NO: 100)

[0875] GD-Hybrid 2 (HB) GD -2): B027 (Fab) αHER2 / GD VH )

[0876] Main chain: VH （曲妥珠单抗） – CH1 – Connector （GQPSG（SEQ ID NO: 45）） – GD VH

[0877] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWVRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 101)

[0878] GD-Halfbody 3 (HB GD -3): B027 (Fab αHER2 / GD VH-杵 )

[0879] Main chain: VH （曲妥珠单抗） – CH1 – linker （GQPSG（SEQ ID NO: 45）） – GD VH-杵

[0880] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 102)

[0881] GD-Halfbody 4 (HB GD -4): B027 (FabαEGFR / GD VL )

[0882] Backbone: VH （西妥昔单抗） – CH1 – linker （GQPSG（SEQ ID NO: 45）） – GD VL

[0883] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQ (SEQ ID NO: 103)

[0884] GD-Halfbody 5 (HB GD -5): B027 (Fab αEGFR / GD VH )

[0885] Backbone: VH （西妥昔单抗） – CH1 – linker （GQPSG（SEQ ID NO: 45）） – GD VH

[0886] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWVRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 104)

[0887] GD-Half body 6 (HB GD -6): B027 (Fab αEGFR / GD VH-杵 )

[0888] Backbone: VH （西妥昔单抗） – CH1 – Linker （GQPSG（SEQ ID NO: 45）） – GD VH-杵

[0889] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 105)

[0890] Fab light chain of trastuzumab

[0891] Light chain: VL （曲妥珠单抗） – CL

[0892] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 69)

[0893] cetuximab Fab light chain

[0894] Light chain: VL （西妥昔单抗） – CL

[0895] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 70)

[0896] B073 Protective Structural Domain Half

[0897] The structure of the B073 protected domain form of the hemimolecule provides information on... Figure 3 A and Figure 3 In A', unlike the unprotected domain form of B036, a single protected domain is attached at its C-terminus to the N-terminus of a second Fc subunit containing the acetamipeloid mutation. The fusion of GD with the Fc subunit is achieved via a short hinge sequence. Through this fusion, GD is localized to the αCD3-VH domain (…). Figure 3 A') or αCD3-VL domain ( Figure 3 A) Adjacent and parallel structures allow for optimal shielding. The following summarizes the use of GD as employed in the examples disclosed herein. VH GD VH-杵 GD VL GD HSAwt Or GD HSA-DIIIThe sequence of the B073 form of the half-molecule. Each half-molecule consists of three polypeptide chains: a first backbone containing the Fab heavy chain of cetuximab, αCD3-SVD, and the first Fc subunit; a second backbone containing the protective domain and the second Fc subunit; and a third polypeptide chain containing the Fab light chain of cetuximab.

[0898] GD-Half-body 7 (HB) GD -7): B073 (Fab) αEGFR / αCD3 VH高 / GD VH )

[0899] Main chain 1: VH （西妥昔单抗） – CH1 – Connector （APAE_10） – VH （αCD3高） -Connector （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0900] (SEQ ID NO: 106)

[0901] Backbone 2: GD VH – linker （铰链区截短） –CH2 （AEASS） – CH3 （臼）

[0902] EVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWVRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 107)

[0903] GD - half - body 8 (HB GD - 8): B073 (Fab αEGFR / αCD3 VH高 / GD VH-杵 )

[0904] Backbone 1: VH （西妥昔单抗） – CH1 – linker （APAE_10） – VH （αCD3高） – linker – CH2 （AEASS） – CH3 （杵）

[0905] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0906] Backbone 2: GD VH-杵 – linker （铰链区截短） –CH2 （AEASS） – CH3 （臼）

[0907] EVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 108)

[0908] GD-Halfbody 9 (HB GD -9): B073 (Fab αEGFR / CD3 VL高 / GD VL )

[0909] Backbone 1: VH （西妥昔单抗） – CH1 – linker （APAE_10） – VL （αCD3高） – linker （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0910] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109)

[0911] Backbone 2: GD VL – Linker （铰链区截短） – CH2 （AEASS） – CH3 （臼）

[0912] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 110)

[0913] GD-Half body 10 (HB GD -10): B073 (Fab αEGFR / CD3 VH高 / GD HSA-DIII )

[0914] Backbone 1: VH （西妥昔单抗） – CH1 – linker （APAE_10） – VH （αCD3高） – linker （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0915] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0916] Backbone 2: GD HSA-DIII – linker （铰链区截短） -CH2 （AEASS） – CH3 （臼）

[0917] LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 111)

[0918] GD-Half body 11 (HB GD -11): B073 (Fab αEGFR / αCD3 VH高 / GD HSAwt )

[0919] Main chain 1: VH （西妥昔单抗） – CH1 – linker （APAE_10） – VH （αCD3高） – linker （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0920] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0921] Backbone 2: GD HSAwt – linker （铰链区截短） -CH2 （AEASS） – CH3 （臼）

[0922] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 112)

[0923] GD - half - body 12 (HB GD - 12): B073 (Fab αEGFR / CD3 VL高 / GD HSAwt )

[0924] Backbone 1: VH （西妥昔单抗） – CH1 – linker（APAE_10） – VL （αCD3高） – linker （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0925] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109)

[0926] Backbone 2: GD HSAwt – linker （铰链区截短） -CH2 （AEASS） – CH3 （臼）

[0927] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 112)

[0928] GD-Hemibody 13 (HB GD -13): B073 (Fab αEGFR / αCD3 VL高 / GD HSA-DIII )

[0929] Backbone 1: VH （西妥昔单抗） – CH1 – linker（APAE_10） – VL （αCD3高） – Connector （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0930] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109)

[0931] Backbone 2: GD HSA-DIII – Connector （铰链区截短） -CH2 （AEASS） – CH3 （臼）

[0932] LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 111)

[0933] Fab light chain of cetuximab:

[0934] Light chain: VL （西妥昔单抗） – CL

[0935] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 70)

[0936] Half body of the B099 protection domain

[0937] The structure of the half body molecule in the B099 form is provided in Figure 3 B and Figure 3In B', unlike the B073 form, the protecting domain and αCD3-SVD are exchanged in such a manner that GD is present on the polypeptide containing the Fab heavy chain and the Fc subunit with the mortar mutation, and αCD3-SVD is present on the polypeptide containing the second Fc subunit with the mortar mutation. Exemplary sequences of CyCAT hemimolecules in the B099 protecting domain form used in the examples of this disclosure are summarized below. Each hemimolecule consists of three polypeptide chains: a first backbone containing the Fab heavy chain of cetuximab, the protecting domain, and the first Fc subunit; a second backbone containing αCD3-SVD and the second Fc subunit; and a third polypeptide chain containing the Fab light chain of cetuximab.

[0938] GD-Hymen 14 (HB) GD -14): B099 (Fab) αEGFR / GD VL / αCD3 VL高 )

[0939] Main chain 1: VH （西妥昔单抗） – CH1 – Connector （APAE_10） – GD VL -Connector （铰链区截短） – CH2 （AEASS） – CH3 （臼）

[0940] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 113)

[0941] Backbone 2: VL （αCD3高） - Linker （铰链区截短） - CH2 （AEASS） – CH3 （杵）

[0942] QSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 114)

[0943] GD-Halfbody 15 (HB GD -15): B099 (Fab αEGFR / GD VH / αCD3 VH高 )

[0944] Backbone 1: VH （西妥昔单抗） – CH1 – Linker （APAE_10） – GD VH – Linker （铰链区截短） – CH2 （AEASS） – CH3 （臼）

[0945] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWVRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 115)

[0946] Backbone 2: VH （αCD3高） - linker （铰链区截短） - CH2 （AEASS） – CH3 （杵）

[0947] EVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 116)

[0948] Fab light chain cetuximab:

[0949] Light chain: VL （西妥昔单抗） – CL

[0950] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 70)

[0951] B077 Protective Structural Domain Half

[0952] The structure of the B077 form of the half-molecule provides for... Figure 3 C and Figure 3 In C', unlike the B039 non-protected domain form, one of the two identical αCD3-SVDs has been replaced by a protected domain, thus requiring the introduction of a mortising mutation in the Fc region. In the B077 form, the single protected domain fuses at its N-terminus with the C-terminus of the second Fab heavy chain, and at its C-terminus with the N-terminus of the Fc region subunit carrying the mortising mutation. Through this fusion, GD is localized to αCD3. SVD The structural domains are adjacent and parallel. The following section summarizes the use of GD. VHGD VH-杵 GD HSAwt Or GD HSA-DIII An exemplary sequence of a half-molecule in the form of the B077 protecting domain. Each half-molecule consists of four polypeptide chains: a main chain containing the Fab heavy chain of cetuximab, αCD3-SVD, and a first Fc region subunit containing a mortar-shaped mutation; a second main chain containing the Fab heavy chain of cetuximab, GD, and a second Fc region subunit containing a mortar-shaped mutation; and two identical polypeptides, each containing the Fab light chain of cetuximab.

[0953] GD-Hymen 16 (HB) GD -16): B077 (Fab) αEGFR / αCD3 VH高 / GD VH )

[0954] Main chain 1: VH （西妥昔单抗） – CH1 – Connector （APAE_10） – VH （CD3高） -Connector （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0955] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0956] Backbone 2: VH （西妥昔单抗） – CH1 – Linker （APAE_10） – αGD VH – CH2 （AEASS） – CH3 （臼）

[0957] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWVRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 117)

[0958] GD-Halfibody 17 (HB GD -17): B077 (Fab αEGFR / αCD3 VH高 / GD VH-杵 )

[0959] Backbone 1: VH （西妥昔单抗） – CH1 – Linker （APAE_10） – VH （αCD3高） – Linker （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0960] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0961] Backbone 2: VH （西妥昔单抗） – CH1 – Linker （APAE_10） – GD （VH-杵） – CH2 （AEASS） – CH3 （臼）

[0962] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 118)

[0963] GD-Half body 18 (HB GD -18): B077 (Fab αEGFR / αCD3 VL高 / GD VL )

[0964] Backbone 1: VH （西妥昔单抗） –CH1–Linker （APAE_10） –VL （αCD3高） –Linker （铰链区截短） –CH2 （AEASS） –CH3 （杵）

[0965] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109)

[0966] Backbone 2: VH （西妥昔单抗） – CH1 – Linker （APAE_10） – GD VL – Linker （铰链区截短） –CH2 （AEASS） – CH3 （臼）

[0967] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 113)

[0968] GD-Halfbody 19 (HB GD -19): B077 (Fab αEGFR / αCD3 VH高 / GD HSAwt )

[0969] Backbone 1: VH （西妥昔单抗） – CH1 – linker （APAE_10） – VH （αCD3高） – linker （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0970] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0971] Backbone 2: VH （西妥昔单抗） – CH1 – Linker （APAE_10） – GD HSAwt – CH2 （AEASS） – CH3 （臼）

[0972]

[0973] GD-half body 20 (HB GD -20): B077 (Fab αEGFR / αCD3 VH高 / GD HSA-DIII )

[0974] Backbone 1: VH （西妥昔单抗） – CH1 – linker （APAE_10） – VH （αCD3高） – linker （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0975] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0976] Backbone 2: VH （西妥昔单抗） – CH1 – linker （APAE_10） – GD HSA-DIII – CH2 （AEASS） – CH3 （臼）

[0977] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 120)

[0978] GD-Half body 21 (HB GD -21): B077 (Fab αEGFR / αCD3 VL高 / GD HSAwt )

[0979] Backbone 1: VH （西妥昔单抗） – CH1 – linker （APAE_10） – VL （αCD3高） – linker （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0980] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109)

[0981] Backbone 2: VH （西妥昔单抗） – CH1 – Linker （APAE_10） – GD （HSAwt） – Linker （铰链区截短） –CH2 （AEASS） –CH3 （臼）

[0982]

[0983] GD - half - body 22 (HB GD -22): B077 (Fab αEGFR / αCD3 VL高 / GD HSA-DIII )

[0984] Main chain 1: VH （西妥昔单抗） – CH1 – linker （APAE_10） – VL （αCD3高） – linker （铰链区截短） – CH2 （AEASS） –CH3 （杵）

[0985] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109)

[0986] Main chain 2: VH （西妥昔单抗） – CH1 – linker （APAE_10） – GD HSA-DIII – linker （铰链区截短） –CH2 （AEASS） –CH3 （臼）

[0987] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCAQPAAPAPAELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 122)

[0988] Fab light chain of cetuximab:

[0989] Light chain: VL （西妥昔单抗） – CL

[0990] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 70)

[0991] B103 Protective Structural Domain Half

[0992] The structure of the B103 form of the half-molecule provides for... Figure 3 D and Figure 3 In D', unlike the B038 unprotected domain form, a single protected domain fuses at its N-terminus with the C-terminus of the second Fc region subunit containing the mortise mutation. Through this fusion, GD is positioned adjacent to and parallel to the αCD3-SVD domain. The use of GD in the examples disclosed herein is summarized below. VH Or GD VL An exemplary sequence of a half-molecule in the form of a B103 protecting domain. Each half-molecule consists of three polypeptide chains: a main chain containing the Fab heavy chain of trastuzumab, a first Fc region subunit containing a mortar-mutated cetyl group, and αCD3-SVD; a second main chain containing a second Fc region subunit containing a mortar-mutated cetyl group and a protecting domain; and two identical polypeptides, each containing the Fab light chain of trastuzumab.

[0993] GD-Half-body 23 (HB) GD -23): B103 (Fab) αHER2 / GD VL / CD3 VL高 )

[0994] Main chain 1: VH （曲妥珠单抗） – CH1 – CH2 （AEASS） – CH3 （杵） - Connector (G4S)4（SEQ ID NO: 46） - VL （CD3高）

[0995] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 86)

[0996] Backbone 2: Linker （铰链区截短） -CH2 （AEASS） – CH3 （臼） - Linker (G4S)4（SEQ ID NO: 46） - GD VL

[0997] KTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQ (SEQ ID NO: 123)

[0998] GD-Halfbody 24 (HB GD -24): B103 (Fab αHER2 / GD VH / αCD3 VH高 )

[0999] Backbone 1: VH （曲妥珠单抗） – CH1 – CH2 （AEASS） – CH3 （杵） - linker (G4S)4（SEQ ID NO: 46） – VH （CD3高）

[1000] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 88)

[1001] Backbone 2: linker （铰链区截短） – CH2 （AEASS） – CH3 （臼） – linker (G4S)4（SEQ ID NO: 46） – GD VH

[1002] KTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWVRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSS (SEQID NO: 124)

[1003] Trastuzumab's Fab light chain:

[1004] Light chain: VL （曲妥珠单抗） – CL

[1005] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 69)

[1006] B101 Protective Structural Domain Half

[1007] The structure of the B101-protected domain form of the hemimolecule provides for the structure of the half-molecule. Figure 3 E and Figure 3 In E', unlike the B064 unprotected domain form, a single GD is fused to the C-terminus of the second Fc region subunit containing the mortise mutation. Through this fusion, the GD is positioned adjacent to and parallel to the αCD3-SVD domain. The use of GD in the examples disclosed herein is summarized below. VH Or GD VLAn exemplary sequence of the B101 form of the hemimolecule. Each hemimolecule consists of four polypeptides: a first backbone comprising a first Fab heavy chain of trastuzumab, a first Fc region subunit containing a mordant mutation, and αCD3-SVD; a second backbone comprising a Fab heavy chain of trastuzumab, a second Fc region subunit containing a mordant mutation, and GD; and two identical polypeptides, each comprising a Fab light chain of trastuzumab.

[1008] GD-Half-body 25 (HB) GD -25): B101 (Fab) αHER2 / GD VL / αCD3 VL高 )

[1009] Main chain 1: VH （曲妥珠单抗） – CH1– CH2 （AEASS） – CH3 （杵） -Connector (G4S)4（SEQ ID NO: 46） – VL （αCD3高）

[1010] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 86)

[1011] Backbone 2: VH （曲妥珠单抗） – CH1 – CH2 （AEASS） – CH3 （臼） – Linker (G4S)4（SEQ ID NO: 46） – GD （VL）

[1012] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQ (SEQ ID NO: 125)

[1013] GD - half - body 26 (HB GD - 26): B101 (Fab αHER2 / GD VH / αCD3 VH高 )

[1014] Backbone 1: VH （曲妥珠单抗） – CH1 – CH2 （AEASS） – CH3 （杵） - linker (G4S)4（SEQ ID NO: 46） - VH （αCD3）

[1015] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 88)

[1016] Backbone 2: VH （曲妥珠单抗） – CH1– CH2 （AEASS） – CH3 （臼） - Linker (G4S)4（SEQ ID NO: 46） - GD （VH）

[1017] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWVRQAPGKGLEWVANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 126)

[1018] Fab light chain of trastuzumab:

[1019] Light chain: VL （曲妥珠单抗） – CL

[1020] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 69)

[1021] General components

[1022] For CyCAT half-molecules employing two distinct backbones, the Fc region is modified by introducing a mutation into the CH3 domain of each Fc subunit using a mortar and pestle technique. This induces heterodimerization between the Fc subunit containing a mutated CH3 domain and another Fc subunit engineered in a complementary manner, containing a different CH3 domain. Additionally, all Fc subunits are modified by introducing a mutation into the CH2 domain to eliminate their mediating effector functions (e.g., the ability to bind ADCC, CDC, ADCP, and / or C1q – referred to herein as the “AEASS” mutation).

[1023] method

[1024] Cloning of CyCAT half molecules

[1025] All nucleic acid sequences or desired gene segments are generated by PCR using appropriate templates, or synthesized internally or by external supplier genes into linear DNA fragments with appropriate flanking regions (e.g., suitable restriction endonuclease recognition sites, adapter sequences). Nucleic acid sequences or gene segments with single restriction endonuclease cleavage sites flanking them are cloned into appropriate mammalian expression vectors using standard molecular biology methods.

[1026] Production of CyCAT half-molecules

[1027] To express the hemisome molecule, exponentially growing eukaryotic HEK293-6E cells were co-transfected with a mammalian expression vector encoding all components of the hemisome molecule, resulting in heteropolymers containing 1:1, 1:1:1, or 1:1:1:1 polypeptide chains. Cell culture supernatants were harvested on day 6 post-transfection and subjected to anti-CH1 affinity chromatography or protein A affinity chromatography, respectively. Buffer was exchanged into 1x DuPont PBS (pH 7.2, Invitrogen), and samples were aseptically filtered (0.2 µm pore size). Protein concentration was determined by UV spectrophotometry, and the purity of the construct was analyzed using CE-SDS (LabChip GXTouch | PerkinElmer | USA) under denaturing, reducing, and non-reducing conditions. UHP-SEC was performed to analyze the hemisome formulation in its native state. The ability of the resulting hemisome to bind to its target antigen was confirmed by standard ELISA using the soluble extracellular domains of human HER2 or human EGFR, respectively.

[1028] Isolation of human T cells

[1029] For example, whole blood from a healthy donor was collected in an S-Monovette container (Sarstedt) containing lithium heparin. 20 mL of blood was transferred to a 50 mL conical tube, mixed with 1 mL of RosetteSep human CD8+ enrichment mixture (Stemcell Technologies, No. 15063), and incubated at room temperature for 20 min. The blood containing the RosetteSep human CD8+ enrichment mixture was diluted with an equal volume of PBS containing 2% fetal bovine serum (Sigma, No. F7524) and 2 mM EDTA. The diluted blood was transferred to a SepMate-50 tube (Stemcell Technologies, No. 85450) containing 15 mL of Lymphoprep density gradient medium (Stemcell Technologies, No. 07811) and centrifuged at 1200 xg for 20 min at room temperature. Transfer the supernatant to a 50 mL conical tube, dilute to 45 mL with PBS containing 2% fetal bovine serum and 2 mMEDTA, and centrifuge at 800 xg for 5 min. Discard the supernatant and resuspend the cell pellet in 1 mL of PBS containing 2% fetal bovine serum. Combine the cell suspensions and transfer to a 50 mL tube, dilute to 30 mL with PBS containing 2% fetal bovine serum. Precipitate the cells by centrifugation at 800 xg for 5 min. Resuspend the cell pellet in 2 mL of 1xPharm Lyse erythrocyte lysis buffer (BD, catalog number 555899) and incubate at 4°C for 10 min. Add PBS containing 2% fetal bovine serum to a final volume of 15 mL. Precipitate the cells at 120 xg for 10 min and decant the supernatant. Wash the cells twice with PBS containing 2% fetal bovine serum and count them (CASY TT apparatus, Beckmann Coulter).

[1030] In vitro cytotoxicity assay

[1031] HER2 / EGFR double-positive SKOV-3 ovarian cancer cells (ATCC) ®HTB-77™ was suspended in medium supplemented with 10% FCS, seeded in black 96-well assay plates (Corning), and incubated overnight at 37°C and 5% CO2 and humidity. The hemimolecules were individually serially diluted in assay medium (RPMI 1640 without phenol red (Gibco, 32404-014); GlutaMAX (Gibco 35050-038); 10% fetal bovine serum) and then mixed in solution to achieve a final CyCAT molecular concentration of 0.00001–1000 nM. CellToxGreen dye (Promega, G8731), serially diluted (and premixed paired) hemimolecules, and purified human T cells (E:T ratio 10:1) were added to the cancer cell lines and incubated at 37°C and 5% CO2 and humidity for 48 or 72 hours. Cytotoxic activity was assessed by measuring the fluorescence of the incorporated CellToxGreen at 485 nm excitation and 535 nm emission using a Tecan Infinite F500 device.

[1032] Example 1: Controlled Experiment

[1033] A) Carrying a single αCD3 VH αCD3 VL GD VH GD VH-杵 Or GD VL The half-molecule of the domain itself does not have the function of binding to CD3 and / or recruiting T cells to kill tumor cells.

[1034] Results of control experiment 1

[1035] Table 4 summarizes the results of Control Experiment 1. As expected, unpaired hemisomes did not mediate SKOV-3 killing in the presence of human T cells (see Table 4; hemisome pairs: 3-12).

[1036] B) Combine the half containing a single αCD3-SVD domain with the half containing a protective domain (GD). VD However, the lack of complementary αCD3-SVD hemisomes does not lead to the formation of functional αCD3-Fv fragments.

[1037] Due to the GD used VH GD VH-杵 and GD VL The domains are derived from a functional αCD3 antibody, therefore the following combinations were further tested.

[1038] a) Hemisomes carrying αCD3-VH and those carrying GD VLhalf-molecule,

[1039] b) Hemisomes carrying αCD3-VL and those carrying GD VH The half of the body, and

[1040] c) Carrying GD VH The half of the body and carrying GD VL half

[1041] Whether this leads to the formation of a functional αCD3-Fv fragment capable of binding to CD3 and redirecting T cells to kill tumor cells. The ability of the tested combination to mediate T cell redirection and kill the SKOV-3 cancer cell line was evaluated as described above.

[1042] Results of control experiment 2

[1043] Table 4 summarizes the results of Control Experiment 2. As expected, the combination of halves that are specific to HER2 and EGFR and carry a pair of complementary αCD3-SVDs resulted in the formation of a functional αCD3-Fv binding fragment and T cell redirection to kill SKOV-3 cells with efficacy in the single picomoles range (see Table 4; half pair: 1 and half pair: 2).

[1044] The half-molecule carrying the αCD3-VL domain and the half-molecule carrying GD VH Or GD VH-杵 The combination of half-molecules carrying the αCD3-VH domain but lacking the complementary αCD3-VH domain does not result in any cytotoxic activity. For half-molecules carrying αCD3-VH and those carrying GD... VL Combinations of the half-molecules yielded the same results. Finally, carrying a single GD... VL half of the body and carrying GD VH Or GD VH-杵 The combination of the half-components also did not show cytotoxic activity. (See Table 2; half-component pairs: 13–22). In summary, the control experiments confirmed that the protective domain disclosed herein cannot form a functional αCD3-Fv binding fragment.

[1045] Table 4: Results of Comparative Experiments 1 and 2

[1046]

[1047] Example 2: Combination of half-molecules lacking protecting domains and half-molecules carrying protecting domains

[1048] The CyCAT method relies on the functional complementarity of the VH domain and the complementary VL domain of the αCD3 antibody on the cell / target, each residing on one of the two complementary CyCAT hemimolecules. However, due to the inherent nature of the association between the complementary antibody variable domains, unwanted residual functional complementarity may occur in solution, in the absence of cells expressing both target antigens, or on cells expressing only one of the two target antigens (e.g., on healthy tissue). Among other factors, such unwanted heterologous association depends on the local concentration of the two complementary hemimolecules and the interfacial affinity between the two complementary αCD3 variable domains.

[1049] Figure 3

[1050] To determine the contribution of unwanted formation (“unwanted heteroassociation”) of the aforementioned functional half-complex to the overall cytotoxic activity of the half-pair in the presence of two target antigens, two parallel assay settings were performed.

[1051] The maximum achievable killing efficacy against SKOV-3 cells (expressing EGFR and HER2) was determined using a conventional “dual-targeting” approach by combining two complementary hemisome pairs, each containing a Fab-targeting motif specific to either HER2 or EGFR and αCD3. VH or αCD3 VL Structural domain.

[1052] To determine the contribution of the CyCAT hemisome complex formed by unwanted heterologous association to the cell-killing activity determined by the dual-targeting approach, the Fab target specificity of a complementary CyCAT hemisome molecule is converted to an irrelevant antigen not present on SKOV-3 cells; in this embodiment of the invention, it is converted to chicken egg lysozyme. This method is referred to herein as the “single-targeting” approach.

[1053] Since one of the hemisomes cannot bind to cancer cell lines, any definite cytotoxic activity can be directly attributed to unwanted heterologous association.

[1054] The ratio of the efficacy measured by the two parallel assay methods (“dual-targeting” and “single-targeting”) defines the assay window or therapeutic window for a given dual-targeting CyCAT half-pair. A larger assay window indicates that the provided half-pair can be mixed or present at higher local concentrations without forming significant amounts of undesirable functional complexes, and thus provides a preferred and particularly safe combination of such half-molecules.

[1055] Results of Example 2

[1056] The experimental results of combining different CyCAT half-body pairs using different half-body forms are summarized in Tables 5–10.

[1057] Generally, and as expected, co-culturing human T cells and EGFR / HER2 double-targeting CyCAT hemisomes in the absence of complementary pairs of molecules lacking any protective domains induces potent killing of SKOV-3 cells in a dose-dependent manner.

[1058] Furthermore, in the presence of the complementary pair of the EGFR / HER2 dual-targeting hemisomes (one of which contains at least one protective domain), co-culturing human T cells and SKOV-3 cells also induced potent killing of SKOV-3 cells in a dose-dependent manner. This result confirms that the presence of the protective domain does not adversely affect the functional complementarity of the αCD3-SVD domain, and also confirms that the presence of the protective domain does not interfere with target binding.

[1059] However, when the target specificity of a hemisomal partner changed from HER2 to lysozyme (single-target), T cell-mediated SKOV-3 cell killing still occurred, albeit less potent than the dual-targeting approach. This result points to the formation of unwanted hemisomal complexes.

[1060] However, surprisingly, in the case of single-target targeting, unwanted cytotoxic activity was significantly reduced when one half-pair also contained a protective domain. These results confirm that the presence of the protective domain effectively inhibits unwanted heterologous association of the complementary CyCAT half-pair molecule in the presence of only one target antigen, but does not interfere with the formation of functional trispecific T cell conjugating antibodies on cells in the presence of two target antigens.

[1061] A half-body result of form B036

[1062] Complementary pairs of dual-targeting halves in the form of B036 (Table 5; half-pair: 1) mediated T-cell killing of SKOV-3 cells with potency in the triple picomolar range. However, the corresponding single-targeting half-pair (half-pair: 2) (where the target specificity changed from HER2 to lysozyme for the first half) still mediated SKOV-3 cell killing with potency in the low double picomolar range, resulting in a 118-fold assay window. Dual-targeting with half-pairs of the non-protective domain form of B036 and the structure-associated B073 (half-pair: 3) or half-pairs of the non-protective domain form of B036 and the protective domain form of B099 (half-pair: 5) showed comparable cytotoxic activity to half-pair: 1, but with significantly larger assay windows (914-fold for half-pair 3 and 1300-fold for half-pair 5). These results clearly demonstrate the ability of the protective domain of the present invention to inhibit unwanted heterologous association in the absence of both target antigens, while not interfering with the desired heterologous association of the half-molecule in the presence of both target antigens.

[1063] Table 5: Cell-killing activity and assay window of half-somatic cells paired with half-somatic cells of the B036 unprotected domain form specific to HER2 or lysozyme and the B036 unprotected domain form or the B073 and B099 protected domain forms specific to EGFR.

[1064]

[1065] Method of determining the assay window ("dual targeting" versus "single targeting") for a given pair of CyCAT halves

[1066] Complementary pairs of dual-targeting hemispheres in the form of B038xB036 unprotected domains (see Table 6; hemisphere pair: 1 and hemisphere pair: 11) mediated SKOV-3 cell killing with potency in the double-digit picomolar range. Corresponding single-targeting hemisphere pairs also mediated cell killing with potency in the double-digit nanomolar range, producing assay windows of 418-fold and 950-fold, respectively.

[1067] By using the variable domain of an inactive antibody as a protective domain, combining the unprotected B038 half with the protected B073 half (see half pairs: 3, 5, and 13) for dual targeting of SKOV-3 cells resulted in cytotoxic activity similar to the aforementioned dual-targeting half in the form of the unprotected B038xB036 half, but with an assay window up to 10-fold larger (see, for example, half pair: 5, assay window 4416-fold).

[1068] Table 6: Cell-killing activity and assay window of half-somatic cells with B038 unprotected domain specific to HER2 or lysozyme paired with half-somatic cells with B036 unprotected domain specific to EGFR or B073 protected domain specific to EGFR.

[1069]

[1070] Interestingly, the use of human serum albumin (HSA-wt) or domain III of human serum albumin (HSA-DIII) resulted in reduced cytotoxic activity and a narrowed assay window for the dual-target hemisphere pair (see hemisphere pairs: 7, 9, 15 and 17, with assay windows of 285, 178, 136 and 421 times, respectively).

[1071] Results for one half in B038 format

[1072] Dual targeting of SKOV-3 cells with hemipants combining the unprotected domains of B038xB063 (see Table 7; hemipants: 1 and 13) or B038xB039 (hemipants: 3 and 15) resulted in cell killing in the low picomolar range, with a assay window ranging from 3565 times that of hemipants 3 to >11000 times that of hemipants 15.

[1073] By utilizing different types of protection structural domains (i.e., GD) VH GD VH-杵 Or GD HSAwt Combining the B038 unprotected domain half with the B077 protected domain half to dual-target SKOV-3 cells produced assay windows ranging from 3324 times that of half pair 20 to, for example, >133.333 times that of half pair 17.

[1074] Table 7: Cell-killing activity and assay window after pairing the half-somatic variant of the B038 unprotected domain, which is specific to HER2 or lysozyme, with the half-somatic variant of the B036 unprotected domain or the B077 protected domain, which is specific to EGFR.

[1075]

[1076] Results for one half in B038 format

[1077] The cytotoxic activity of the B064 unprotected domain half-somatic pairing with the B073 protected domain half-somatic pairing, or the B064 half-somatic pairing with the B099 protected domain half-somatic pairing (including inactive GD) VH Inactive GD VH-杵 or inactive GD VLThe cytotoxic activity of the half-paired haploids was comparable to that of the half-paired haploids of the B064 form and the half-paired haploids of the B036 unprotected domain form, but the assay windows were significantly larger (see Table 8; half-pairs: 3, 5, 9, 17, and 23). The assay windows for these half-pairs ranged from 6104 times that of half-pair 23 to >40,000 times that of half-pair 3.

[1078] Results for one half in B064 format Exemplary results of cytotoxicity assays for half-pairs 1 and 2 and half-pairs 3 and 4, as determined according to Table 8, are shown.

[1079] Table 8: Cell-killing activity and assay window of half-somatic cells in the form of B064 (specific to HER2 or lysozyme) paired with half-somatic cells in the forms of B036, B073, or B099 (specific to EGFR).

[1080]

[1081] Figure 5 Results for one half in B064 format Results for one half in B064 format

[1082] The cytotoxic activity of the B064 unprotected domain half-socket paired with the B077 protected domain half-socket is comparable to that of the dual-targeting half-socket pairs of B064xB039 or B064xB063 unprotected domain half-sockets (see Table 9).

[1083] However, it can be observed that when using GD, compared to the measurement window of the corresponding half-pairs (e.g., half-pairs: 1, 11, 17, and 19) without utilizing any protective structural domains, VH GD VH-杵 Or GD VL The determination window for half-pairs of the protective structural domains (e.g., half-pairs 13, 15, 19, and 21, as shown in Table 9) is significantly larger.

[1084] Table 9: Cell-killing activity and assay window of half-somatic cells with B064 unprotected domain form specific to HER2 or lysozyme paired with half-somatic cells with B039 or B063 unprotected domain form or B077 protected domain form specific to EGFR.

[1085]

[1086] A half-body result in the form of B050

[1087] The cytotoxic activity of the B050 unprotected domain form half-mouse paired with the B073 or B077 protected domain form half-mouse is comparable to that of the associated dual-targeting half-mouse pairs of B050xB036 or B050xB039 unprotected domain form half-mouse (see Table 10). However, the assay windows for half-mouse pairs utilizing protected domains (e.g., for half-mouse pairs: 1, 7, 13, and 19, as shown in Table 10) are significantly larger than those for corresponding half-mouse pairs that do not utilize any protected domain (e.g., half-mouse pairs: 1, 7, 13, and 19).

[1088] Table 10: Cell-killing activity and assay window of half-somatic cells with B050 non-protective domain specific to HER2 or lysozyme paired with half-somatic cells with B036 or B039 non-protective domain specific to EGFR or B073 or B077 protective domain specific to EGFR.

[1089] .

Claims

1. A pair of complementary half-molecules, which contain a) The first half-molecule (HB1) contains i. The first Fab fragment (Fab1) that is specific to the first antigen (AG1). ii. The first VH (αCD3-VH1) or first VL (αCD3-VL1) of the CD3-specific Fv fragment (αCD3-Fv), and iii. The first protective structural domain (GD1), and b) The second hemimolecule (HB2) contains i. The second Fab fragment (Fab2) is specific to the second antigen (AG2). ii. The complementary first VH (αCD3-VH2) or complementary first VL (αCD3-VL2) of the αCD3-Fv, and iii. Optional second protective structural domain (GD2). HB1 and HB2 are not connected by covalent bonds. HB1 and HB2 can form heterodimers. The formation of the heterodimers of HB1 and HB2 occurs via dimerization of αCD3-VH1 and αCD3-VL2 or dimerization of αCD3-VL1 and αCD3-VH2. The dimerization of αCD3-VH1 and αCD3-VL2 or the dimerization of αCD3-VL1 and αCD3-VH2 leads to the formation of αCD3-Fv, and The heterodimerization of HB1 and HB2 leads to the formation of T-cell binding trispecific antibodies.

2. The pair of complementary half-molecules according to claim 1, wherein GD1 is not bound to αCD3-VH1, αCD3-VL1 or HB1, and wherein GD2 is not bound to αCD3-VH2, αCD3-VL2 or HB2.

3. A pair of complementary hemimolecules according to any one of the preceding claims, wherein each of GD1 and GD2 is a soluble protein, a soluble polypeptide, a soluble globular protein, or a soluble globular polypeptide.

4. A pair of complementary half-molecules according to any one of the preceding claims, wherein each of GD1 and GD2 has a molecular size of less than 70 kDa.

5. A pair of complementary hemimolecules according to any one of the preceding claims, wherein each of GD1 and GD2 is selected from the group consisting of albumin, fibrinogen, fibronectin, hemoglobin, transferrin, immunoglobulin domains or fragments thereof.

6. A pair of complementary half-molecules according to any one of the preceding claims, wherein if HB1 contains αCD3-VH1, then GD1 can be selected as the VH domain (GD... VH Instead of VL domain (GD) VL Furthermore, if HB1 contains αCD3-VL1, then GD1 can be selected as the VL domain (GD...). VL Instead of the VH domain (GD) VH Furthermore, if HB2 contains αCD3-VH2, then GD2 can be selected as a VH domain (GD...). VH Instead of VL domain (GD) VL Furthermore, if HB2 contains αCD3-VL2, then GD2 can be selected as a VL domain (GD...). VL Instead of the VH domain (GD) VH ).

7. A pair of complementary hemimolecules according to any one of the preceding claims, wherein each of GD1 and GD2 is not released from HB1 or HB2, respectively, after administration to a subject.

8. A pair of complementary hemimolecules according to any one of the preceding claims, wherein each of GD1 and GD2 cannot be cleaved from HB1 or HB2 by a tumor-specific protease, respectively.

9. A pair of complementary half-molecules according to any one of the preceding claims, wherein GD1 and αCD3-VH1 or GD1 and αCD3-VL1 are positioned adjacent to each other and parallel, and wherein GD2 and αCD3-VH2 or GD2 and αCD3-VL2 are positioned adjacent to each other and parallel.

10. A pair of complementary half molecules according to any one of the preceding claims, wherein GD1 and αCD3-VH1 or GD1 and αCD3-VL1 of HB1 are present on two different polypeptides, and wherein GD2 and αCD3-VH2 or GD2 and αCD3-VL2 of HB2 are present on two different polypeptides.

11. A pair of complementary half-molecules according to any one of the preceding claims, wherein GD1 is present on HB1 in the absence of AG1 and / or AG2. i. Inhibit the dimerization of αCD3-VH1 and αCD3-VL2, or the dimerization of αCD3-VL1 and αCD3-VH2, respectively. ii. Inhibit the formation of αCD3-Fv, iii. Inhibit the dimerization of HB1 and HB2, and / or iv. Inhibit the formation of the T cell-conjugated trispecific antibodies.

12. A pair of complementary half-molecules according to any one of the preceding claims, wherein GD1 is present in the presence of AG1 and AG2. i. Does not inhibit the binding of Fab1 to AG1 or the binding of Fab2 to AG2. ii. Does not inhibit the dimerization of αCD3-VH1 and αCD3-VL2 or the dimerization of αCD3-VL1 and αCD3-VH2. iii. Does not inhibit the formation of αCD3-Fv, iv. Does not inhibit the binding of αCD3-Fv to CD3, v. Does not inhibit the dimerization of HB1 and HB2. vi. Does not inhibit the formation of the T cell-conjugated trispecific antibody, and / or vii. The activity of the trispecific antibodies-mediated T cell redirection killing of cells with AG1 and AG2 on their cell surface is not inhibited.

13. A pair of complementary hemimolecules according to any one of the preceding claims, wherein the activity of the trispecific antibody-mediated T cell redirection killing of cells having AG1 and AG2 on their cell surface is at least twice that of the cells having AG1 or AG2 on their cell surface.

14. A pair of complementary hemimolecules according to any one of the preceding claims, wherein, in the presence of GD1 on HB1 or GD2 on HB2, the IC50 measured against hemimolecule-pair-induced T cell-mediated killing of cells expressing AG1 or AG2 on their cell surface is... 50 The concentration and IC50 were determined by measuring the T-cell-mediated killing of cells expressing AG1 and AG2 on their cell surface in response to the half-body. 50 The concentration ratio was increased compared to the same ratio measured for a pair of complementary half-molecules lacking GD1 on HB1 or GD2 on HB2.

15. The pair of complementary half-molecules according to claim 14, wherein the IC 50 The concentration ratio increases by at least 1.5 times.

16. A pair of complementary half-molecules according to any one of the preceding claims, wherein HB1, HB2, αCD3-VH1, αCD3-VH2, αCD3-VL1 and αCD3-VL2 cannot bind to CD3 themselves.

17. A pair of complementary half-molecules according to any one of the preceding claims, wherein AG1 and AG2 are present on the surface of the same cell.

18. A pair of complementary half-molecules according to any one of the preceding claims, wherein HB1 further comprises a first Fc region, and / or wherein HB2 further comprises a second Fc region.

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