Combination of antigen-binding molecules

Abstract

The present disclosure provides a combination or set of two antigen-binding molecules, particularly an asymmetric combination of such antigen-binding molecules. Each of the two antigen-binding molecules is composed of a targeting moiety with specificity for a tumor-associated antigen fused to either the VL or VH domain of an antibody Fv domain specific for CD3. When the two antigen-binding molecules bind to their target antigens on the cell surface, the complementary VL and VH domains can bind to each other, thereby reconstituting a functional CD3-specific Fv domain and non-covalently dimerizing the two antigen-binding molecules. The trispecific heterodimeric antibody molecule thus formed on-cell can induce and stimulate cytotoxic T cells for tumor cell destruction.

Description

[Technical field]

[0001] The present disclosure relates to combinations or sets of antigen binding molecules, which are preferably similar and activated on-cell. The trispecific heteromeric antibody molecules thus formed on-cell can induce and stimulate immune cells for tumor cell destruction. The asymmetric combination approach described herein is particularly useful for targeting different epitopes on tumor-associated antigens, thereby improving tumor cell killing. [Background technology]

[0002] Typically, therapeutic concepts based on the use of bispecific antibodies rely on the recruitment of effector cells, thus targeting, on the one hand, tumor-associated antigens (TAA) and, on the other hand, CD3, a proven T cell stimulating antigen with therapeutic relevance. In these concepts, target cells (e.g. tumor cells) and T cells are cross-linked via bispecific antibodies, forming an immune synapse, which allows the effector T cells to directly kill the target cells [Miller and Kontermann,Bispecific antibodies for cancer immunotherapy:Current perspectives.BioDrugs 2010,24(2):89-98]. Since the immune synapse depends on the distance from the target cell to the effector T cell, it has been proposed that the target epitope on the tumor cell and CD3 on the T cell must be in close proximity to enhance the killing effect (Bluemel C.,Cancer Immunol.Immunother.2010 Aug;59(8):1197-209). It has also been suggested that targeting near-cell surface epitopes of tumor-associated antigens may also contribute to more efficient T cell redirected killing. In general, it appears that the closer effector T cells are redirected to the tumor cell surface, the more efficiently they can destroy the tumor cells.

[0003] One major problem in CD3-targeted therapy is the challenge of toxicity due to off-target T cell activation. Such dose-limiting toxicity is essentially determined by the inherent ability of the CD3-binding domain to stimulate T cells regardless of the presence of target cells. Therefore, to reduce such dose-limiting toxicity, monovalent binding to CD3 with moderate binding affinity has been established. Another approach discussed in the art to reduce the toxicity of CD3-cobinding antibodies is the use of dual targeting of two different TAAs on tumor cells. Such dual targeting leads to improved selectivity against normal tissues that express both target antigens only one at a time or at low levels. However, conventional multispecific antibodies developed to bind to such a combination of two TAAs may result in high off-target effects, since binding to only one of the two TAA antigens may still be sufficient to recruit immune cells for the destruction of cells expressing either target molecule.

[0004] Another drawback of the conventional multispecific antibody formats of the prior art is that they are usually produced as one complete and structurally fixed molecule, with the distance between each tumor targeting domain and the CD3 binding domain being almost the same.The constant distance in such molecules cannot take into account the various distances of the target antigens or antigen epitopes.As outlined above, the effectiveness and potency of T cell-inducing bispecific antibodies targeting two different antigens can be highly dependent on the target epitopes, for example, their relative positions to each other and / or their relative positions to the cell membrane.

[0005] Thus, there is a need to provide improved multispecific antibody formats that address the competing needs of efficacy and off-target toxicity in CD3-based immunotherapeutics.

[0006] One approach that overcomes some of the aforementioned drawbacks is described in WO 2013 / 104804 [JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG] and is called the "DUAL ANTIGEN-INDUCED BIPARTITE FUNCTIONAL COMPLEMENTATION" or "HEMIBODY® approach". As part of the aforementioned approach, two polypeptides are designed, each consisting of a targeting moiety (e.g., a single chain variable fragment (scFv) or an antibody Fab fragment) fused to either the variable light domain (VL) or the complementary variable heavy domain (VH) of a split or dissociated T cell activating anti-CD3 antibody Fv domain. Unpaired VH and VL domains (e.g., split CD3 VH and split CD3 VL domains) cannot bind to CD3 alone. However, when the two polypeptides bind to a target antigen on the cell surface via the targeting moiety, the complementary VL and VH domains come into close proximity and interact with each other to reconstitute the original functional CD3Fv domain. The trispecific heterodimeric antibody thus formed on-target cell (as in the conventional trivalent trispecific antibody format) induces and stimulates T cells for the destruction of cancer cells.

[0007] Since the initial publication, the HEMIBODY® approach has undergone further refinements to address pharmacokinetic issues, i.e., product homogeneity and unwanted residual hetero-association of Hemibodies in solution in the absence of target cells (WO 2016 / 023909, WO 2017 / 087789, WO 2019 / 077092, WO 2020 / 216883, WO 2020 / 216879, WO 2020 / 216878, WO 2020 / 223108, WO 2020 / 010104). Summary of the Invention [Problem to be solved by the invention]

[0008] However, none of the known approaches in the art provide tailor-made antigen-binding molecules optimized for combinatorial on-cell multispecific antibody formation that takes into account the various distances afforded by the targeted epitopes, enabling superior T cell-mediated tumor cell killing. [Means for solving the problem]

[0009] In view of the above-mentioned needs, it is an object of the present disclosure to provide a set of antigen-binding molecules suitable for binding to two target antigens or antigen epitopes (such as tumor-associated antigens) on a target cell and one antigen on an immune effector cell, such as CD3. Accordingly, the present disclosure provides an on-cell forming trispecific antibody composed of a set of antigen-binding molecules as disclosed herein.

[0010] Further provided herein is a combination or set of a first antigen-binding molecule and a second antigen-binding molecule, wherein each of the two antigen-binding molecules is composed of a targeting moiety having specificity for a target antigen fused to either an unpaired VL or VH domain of an antibody Fv domain specific for a third antigen, preferably human CD3, and the two antigen-binding molecules are not linked by a covalent bond.

[0011] The unpaired VL or VH domains present in the antigen-binding molecules of the present disclosure cannot bind to their targets, such as CD3, alone. However, when both antigen-binding molecules bind to their target antigens expressed on the surface of the same cell, the complementary unpaired VL and VH domains come into close proximity and interact with each other to reconstitute the original anti-CD3 antibody Fv domain, thus providing an on-cell forming trispecific heterodimeric antibody that can induce and stimulate T cells for the destruction of cancer cells.

[0012] The present disclosure also relates to specific combinations or sets of such first and second antigen-binding molecules that differ in shape or structure and in the presence or absence of individual components (such as IgG Fc regions). In particular, the antigen-binding molecules included in the set of antigen-binding molecules disclosed herein may differ in the distance of the targeting moiety to the unpaired variable domain, such as the unpaired VH or VL domain specific for CD3. A specific combination of antigen-binding molecules disclosed herein may result in a symmetric on-cell forming trispecific antibody. In such a symmetric combination, the distance of each targeting moiety to the newly formed anti-CD3 Fv domain is approximately the same. Preferably, a specific combination or set of antigen-binding molecules disclosed herein results in an asymmetric on-cell forming trispecific antibody. In such an asymmetric combination, the distance of each targeting moiety to the newly formed anti-CD3 Fv domain is significantly different.

[0013] The inventors of the present disclosure have surprisingly found that such asymmetric combinations are particularly useful for targeting different antigens or antigen epitopes on target cells, thereby resulting in superior killing of the target cells, which was unexpected since it was thought that functional complementation of unpaired VH and VL domains (e.g., with specificity for CD3) would likely not occur due to the aforementioned divergent distances.

[0014] In one embodiment, the present disclosure provides a set of antigen binding molecules consisting of or comprising a first and a second antigen binding molecule, wherein the first and second antigen binding molecules are selected based on a first antigen or a first antigen epitope and a second antigen or a second antigen epitope, e.g., according to their expression levels.

[0015] In one embodiment of the present disclosure, the first and second antigen binding molecules are selected based on the distance from the first target antigen epitope to the cell surface and the distance from the second target antigen epitope to the cell surface.

[0016] In one embodiment of the present disclosure, the first and second antigen binding molecules are selected based on the distance from the first antigen or first antigen epitope to the second antigen or second antigen epitope.

[0017] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first targeting moiety comprising a first binding site specific for a first antigen; ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. A first antigen-binding molecule consisting of or comprising: a first antigen-binding molecule in which a first targeting moiety is fused to the N-terminus of either the VH or VL domain of the second binding site via a first peptide linker; b) from its N-terminus to its C-terminus: i. a second targeting moiety comprising a third binding site specific for a third antigen; ii. a second peptide linker; iii. a first Fc region composed of a first and a second Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; iv. a third peptide linker, and v. A complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: a second targeting moiety is fused to the N-terminus of the first Fc region subunit via a second peptide linker; the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a third peptide linker; and A second antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a fourth peptide linker. The present invention provides a set of antigen-binding molecules consisting of or comprising:

[0018] In one embodiment, an antigen binding molecule according to the present disclosure comprises only one of the variable domains of the second binding site.

[0019] In an embodiment of the present disclosure, the first antigen binding molecule optionally further comprises: i. a fifth peptide linker; and ii. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; wherein: the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of a third Fc region subunit via a fifth peptide linker; The N-terminus of the fourth Fc region subunit is fused to a sixth peptide linker.

[0020] In one embodiment of the present disclosure, the first antigen binding molecule further comprises: i. a fifth peptide linker; ii. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; wherein: the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of a third Fc region subunit via a fifth peptide linker; The N-terminus of the fourth Fc region subunit is fused to a sixth peptide linker.

[0021] In one embodiment of the disclosure, the second antigen binding molecule optionally further comprises: i. a third targeting moiety comprising a fourth binding site specific for a third antigen. consisting of or comprising Here, a third targeting moiety is fused to the N-terminus of the second Fc region subunit of the second antigen-binding molecule subunit via a fourth peptide linker.

[0022] In one embodiment of the disclosure, the second antigen binding molecule further comprises: i. a third targeting moiety comprising a fourth binding site specific for a third antigen. consisting of or comprising Here, a third targeting moiety is fused to the N-terminus of the second Fc region subunit via a fourth peptide linker.

[0023] In one embodiment of the present disclosure, the second targeting moiety and the third targeting moiety are the same. In one embodiment, the fourth peptide linker is the same as the second peptide linker.

[0024] In one embodiment of the disclosure, the targeting moiety is an antibody or antibody fragment. In one embodiment of the disclosure, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment of the disclosure, the first and / or second and / or third targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab. In one embodiment, the first and second targeting moieties are Fabs. In one embodiment, the first, second and third targeting moieties are Fabs. In one embodiment, the first targeting moiety is a first Fab, the second targeting moiety is a second Fab, and the third targeting moiety is a third Fab. In one embodiment, the second binding site is an Fv domain.

[0025] In one embodiment, the C-terminus of the first Fab heavy chain is fused to the N-terminus of either the VH or VL domain of the second binding site of the first antigen binding molecule via a first peptide linker. In one embodiment, the C-terminus of the second Fab heavy chain is fused to the N-terminus of the first Fc region subunit of the second antigen binding molecule via a second peptide linker. In one embodiment, the C-terminus of the third Fab heavy chain is fused to the N-terminus of the second Fc region subunit via a fourth peptide linker.

[0026] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first Fab comprising a first binding site specific for a first antigen; ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. A first antigen-binding molecule consisting of or comprising: A first antigen-binding molecule in which the C-terminus of a first Fab heavy chain is fused to the N-terminus of either the VH or VL domain of a second binding site via a first peptide linker; b) The following: i. a second Fab comprising a third binding site specific for a third antigen; ii. a second peptide linker; iii. a first Fc region composed of a first and a second Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; iv. a third peptide linker, and v. The complementary VH or VL domain of the second binding site In the second antigen-binding molecule, which consists of or comprises: the C-terminus of the second Fab heavy chain is fused to the N-terminus of the first Fc region subunit via a second peptide linker; the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a third peptide linker; and A second antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a fourth peptide linker. The present invention provides a set of antigen-binding molecules consisting of or comprising:

[0027] In one embodiment of the present disclosure, the first antigen binding molecule optionally further comprises: i. a fifth peptide linker; and ii. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; wherein: the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of a third Fc region subunit via a fifth peptide linker; The N-terminus of the fourth Fc region subunit is fused to a sixth peptide linker.

[0028] In one embodiment of the present disclosure, the first antigen binding molecule further comprises: i. a fifth peptide linker; and ii. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; wherein: the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of a third Fc region subunit via a fifth peptide linker; The N-terminus of the fourth Fc region subunit is fused to a sixth peptide linker.

[0029] In one embodiment of the disclosure, the second antigen binding molecule optionally further comprises: i. a third Fab comprising a fourth binding site specific for a third antigen. wherein: Here, the C-terminus of the third Fab heavy chain is fused to the N-terminus of the second Fc region subunit via a fourth peptide linker.

[0030] In one embodiment of the disclosure, the second antigen binding molecule further comprises: i. a third Fab comprising a fourth binding site specific for a third antigen. wherein: Here, the C-terminus of the third Fab heavy chain is fused to the N-terminus of the second Fc region subunit via a fourth peptide linker.

[0031] In one embodiment of the present disclosure, the first antigen-binding molecule consists of or comprises a first and a second polypeptide, wherein: a) the first polypeptide comprises a light chain of a first Fab; b) the second polypeptide comprises from its N-terminus to its C-terminus: i. a heavy chain of a first Fab; ii. a first peptide linker; and iii. either the VH domain or the VL domain of a second binding site specific for a second antigen. Includes.

[0032] In one embodiment of the present disclosure, the first antigen-binding molecule consists of or comprises a first, second and third polypeptide, wherein: a) the first polypeptide comprises a light chain of a first Fab; b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. Fifth peptide linker v. a third Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; and c) the third polypeptide comprises from its N-terminus to its C-terminus: i. a sixth peptide linker; and ii. from its N-terminus to its C-terminus, a fourth Fc region subunit composed of a CH2 and a CH3 domain. Includes.

[0033] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a fourth, fifth and sixth polypeptide, wherein: a) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. a fourth peptide linker, ii. a second Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus Including, b) the fifth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a second peptide linker; iii. a first Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; iv. a third peptide linker; v. The complementary VH or VL domain of the second binding site and c) the sixth polypeptide comprises the light chain of the second Fab.

[0034] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a fourth, fifth, sixth and seventh polypeptide, wherein: a) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a third Fab; ii. a fourth peptide linker; iii. a second Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; Including, b) the fifth polypeptide comprises from its N-terminus to its C-terminus: i. The heavy chain of the second Fab ii. A second peptide linker iii. a first Fc region subunit composed of, from its N-terminus to its C-terminus, a CH2 and a CH3 domain; iv. A third peptide linker v. The complementary VH or VL domain of the second binding site Including, c) the sixth polypeptide comprises a light chain of a second Fab, and d) the seventh polypeptide comprises the light chain of the third Fab.

[0035] In one embodiment, the antigen-binding molecule of the present disclosure has a structure as depicted in FIG. 1A, FIG. 1B, or FIG. 1C, or FIG. 4A. In one embodiment, the first antigen-binding molecule of the present disclosure has a structure as depicted in FIG. 1A. In one embodiment, the first antigen-binding molecule of the present disclosure has a structure as depicted in FIG. 1B. In one embodiment, the second antigen-binding molecule of the present disclosure has a structure as depicted in FIG. 1C. In one embodiment, the second antigen-binding molecule of the present disclosure has a structure as depicted in FIG. 4A. In one embodiment, the present disclosure provides a set of antigen-binding molecules, wherein the first antigen-binding molecule has a structure as depicted in FIG. 1A or 1B, and wherein the second antigen-binding molecule has a structure as depicted in FIG. 1C or FIG. 4A. In one embodiment, the set of antigen-binding molecules of the present disclosure has a structure as depicted in FIG. 2A-2F and FIG. 4B-4. In one embodiment, the set of antigen-binding molecules of the present disclosure has a structure as depicted in FIG. 2F. In one embodiment, the set of antigen-binding molecules according to the present disclosure has a structure as shown in Figures 2D-2F and Figures 4C and 4D.

[0036] In an embodiment of the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, in the set of antigen-binding molecules, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond.

[0037] In one embodiment, the VH and VL domains of the second binding site can be non-covalently associated, thereby forming the second binding site. In one embodiment, the VH and VL domains of the second binding site can be non-covalently associated with each other, thereby forming the second binding site.

[0038] In one embodiment, either the VH or VL domain of the second binding site of the first antigen-binding molecule and the complementary VH or VL domain of the second binding site of the second antigen-binding molecule can be non-covalently bound to each other, thereby forming a second binding site. In one embodiment, either the VH or VL domain of the second binding site of the first antigen-binding molecule and the complementary VH or VL domain of the second binding site of the second antigen-binding molecule can be non-covalently bound to each other, thereby forming a second binding site.

[0039] In one embodiment, the non-covalent binding results in functional complementarity between either the VH or VL domain of the second binding site of the first antigen-binding molecule and the complementary VH or VL domain of the second binding site of the second antigen-binding molecule. In an embodiment of the present disclosure, a functional second binding site is formed upon non-covalent binding of either the VH or VL domain of the second binding site of the first antigen-binding molecule and the complementary VH or VL domain of the second binding site of the second antigen-binding molecule.

[0040] In one embodiment, the non-covalent binding occurs in solution. In one embodiment, the non-covalent binding occurs when the first and second antigen-binding molecules bind to their target antigens via their targeting moieties. In one embodiment, the non-covalent binding occurs when the first and second antigen-binding molecules bind to target antigens present on the same cell. In one embodiment of the present disclosure, the first and second antigen-binding molecules do not bind in the absence of cells expressing the first and third antigens on their cell surface. In one embodiment of the present disclosure, the first and second antigen-binding molecules do not bind in the absence of the first and third antigens.

[0041] In one embodiment, non-covalent binding of the VH and VL domains of the second binding site dimerizes the first and second antigen-binding molecules. In one embodiment, the first antigen-binding molecule and the second antigen-binding molecule according to the present disclosure can form a heteromeric molecule. In one embodiment, the heteromeric molecule is formed by binding of the VH or VL domain of the second binding site of the first antigen-binding molecule with the complementary VH or VL domain of the second binding site of the second antigen-binding molecule.

[0042] In one embodiment, the amount of heteromeric molecules formed in the presence of cells expressing the first and third antigens is greater than in the absence of such cells or in the presence of cells expressing either the first or third antigen. In one embodiment, the amount of heteromeric molecules formed in the presence of the first and third antigens is greater than in the absence of both antigens or in the presence of either the first or third antigen. In one embodiment, the heteromeric molecule is a trispecific antibody molecule.

[0043] In an embodiment of the disclosure, the non-covalent association of the VH and VL domains of the second binding site occurs selectively in the presence of a cell expressing the first antigen and a third antigen, in one embodiment of the disclosure, the non-covalent association of the VH and VL domains of the second binding site occurs selectively in the presence of the first antigen and a third antigen.

[0044] In one embodiment of the present disclosure, the ability of the heteromeric molecule formed between the first antigen-binding molecule and the second antigen-binding molecule to redirect T cell-mediated cell killing is higher in the presence of cells expressing the first and third antigens than in the absence of such cells or in the presence of cells expressing only the first antigen or the third antigen.

[0045] In one embodiment of the present disclosure, the second binding site is an Fv domain or Fv region. In one embodiment, the second binding site is an Fv domain or Fv region specific for CD3. In one embodiment, the VH domain of the Fv domain is present on the first antigen binding domain and the complementary VL domain of the Fv domain is present on the second antigen binding domain, or vice versa.

[0046] In one embodiment of the present disclosure, when the second binding site of the first antigen binding molecule comprises or is a VH domain, the second binding site of the second antigen binding molecule is or comprises a complementary VL domain; alternatively, when the second binding site of the first antigen binding molecule comprises or is a VL domain, the second binding site of the second antigen binding molecule comprises or is a complementary VH domain.

[0047] In one embodiment of the present disclosure, the first antigen-binding molecule comprises either the VH or VL domain of the second binding site, but does not comprise both antibody variable domains. In one embodiment of the present disclosure, the second antigen-binding molecule comprises either the VH or VL domain of the second binding site, but does not comprise both antibody variable domains.

[0048] In one embodiment of the disclosure, the VH and VL domains of the second binding site are derived from the same antibody.

[0049] In one embodiment of the present disclosure, binding of the second binding site to the second antigen is stronger in the presence of cells expressing the first antigen and the third antigen than in the absence of such cells or in the presence of cells expressing only the first antigen or the third antigen.

[0050] In one embodiment of the present disclosure, binding of the second binding site to the second antigen is stronger in the presence of the first antigen and the third antigen than in the absence of the first antigen and the third antigen, or in the presence of only the first antigen or the third antigen.

[0051] In one embodiment of the present disclosure, the VH alone or the VL alone of the second binding site cannot bind to the second antigen. In one embodiment of the present disclosure, the first antigen-binding molecule alone cannot bind to the second antigen. In one embodiment of the present disclosure, the second antigen-binding molecule alone cannot bind to the third antigen. In one embodiment of the present disclosure, neither the first antigen-binding molecule nor the second antigen-binding molecule alone can bind to the second antigen. In one embodiment of the present disclosure, the first antigen-binding molecule alone cannot redirect T cell-mediated killing of a cell. In one embodiment of the present disclosure, the second antigen-binding molecule alone cannot redirect T cell-mediated killing of a cell.

[0052] In one embodiment, in the set of antigen-binding molecules according to the present disclosure, the length of the peptide linker is selected from those having a length of 5 to 50 amino acid residues, preferably 5 to 29 amino acid residues. In one embodiment, in the set of antigen-binding molecules according to the present disclosure, the peptide linker has a length of 5 to 49 amino acid residues, preferably 5 to 29 amino acid residues.

[0053] In one embodiment, the peptide linker is selected from those having a length of 5, 20 or 29 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 5 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 20 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 29 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 5, 9, 10, 15, 19, 20, 25, 29, 40, 45 or 49 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 5, 9, 10, 15, 19, 20, 25, 29, 40, 45 or 49 amino acid residues. In one embodiment, a peptide linker according to the present disclosure comprises only naturally occurring amino acid residues. In one embodiment, a peptide linker according to the present disclosure comprises only naturally occurring amino acid residues, but does not comprise cysteine.

[0054] In one embodiment, in the set of antigen-binding molecules according to the present disclosure, the length of the first and third peptide linkers is selected from those having a length of 5 to 40 amino acid residues, preferably 5 to 20 amino acid residues, and more preferably 5 and / or 20 amino acid residues. In one embodiment, in the set of antigen-binding molecules according to the present disclosure, the length of the first and third peptide linkers is selected from those having a length of 10 to 45 amino acid residues.

[0055] In one embodiment, the first peptide linker has a length of 5 to 40 amino acid residues. In one embodiment, the first peptide linker has a length of 5 to 40 amino acid residues, preferably 5 to 20 amino acid residues. In one embodiment, the first peptide linker has a length of 5 amino acid residues. In one embodiment, the first peptide linker has a length of 10 amino acid residues. In one embodiment, the first peptide linker has a length of 20 amino acid residues. In one embodiment, the first peptide linker has a length of 40 amino acid residues. In one embodiment, the first peptide linker has a length of 5, 10, 20 or 40 amino acid residues.

[0056] In one embodiment, the first peptide linker has a length of 5 to 45 amino acid residues. In one embodiment, the first peptide linker has a length of 10 to 45 amino acid residues. In one embodiment, the first peptide linker has a length of 10 to 45 amino acid residues, preferably 10 to 25 amino acid residues. In one embodiment, the first peptide linker has a length of 10 amino acid residues. In one embodiment, the first peptide linker has a length of 15 amino acid residues. In one embodiment, the first peptide linker has a length of 25 amino acid residues. In one embodiment, the first peptide linker has a length of 45 amino acid residues. In one embodiment, the first peptide linker has a length of 10, 15, 25 or 45 amino acid residues.

[0057] In one embodiment, the first peptide linker consists only of naturally occurring amino acid residues. In one embodiment, the first peptide linker contains only naturally occurring amino acid residues but does not contain cysteines.

[0058] In one embodiment, the first peptide linker comprises the amino acid sequence of: GQPSG (SEQ ID NO: 35). In one embodiment, the first peptide linker comprises the amino acid sequence of: AQPAAPAPAE (SEQ ID NO: 51). In one embodiment, the first peptide linker comprises the amino acid sequence of: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16). In one embodiment, the first peptide linker comprises the amino acid sequence of: AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 31). In one embodiment, the first peptide linker comprises the amino acid sequence of: AQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 52). In one embodiment, the first peptide linker comprises the amino acid sequence of: GQPSG (SEQ ID NO: 35) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16). In one embodiment, the first peptide linker is selected from the group consisting of the amino acid sequences of the following: GQPSG (SEQ ID NO: 35), AQPAAPAPAE (SEQ ID NO: 51), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16), AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 31) and AQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 52). In one embodiment, the first peptide linker further comprises the amino acid sequence of EPKSC (SEQ ID NO: 100) at its C-terminus.

[0059] In one embodiment, the first peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105. In one embodiment, the first peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105. In one embodiment, the first peptide linker comprises any of the amino acid sequences as shown in Table 22. In one embodiment, the first peptide linker does not include an Fc region.

[0060] In one embodiment, the third peptide linker has a length of 5 to 20 amino acid residues, preferably 20 amino acid residues. In one embodiment, the third peptide linker has a length of 20 amino acid residues. In one embodiment, the third peptide linker has a length of 5 amino acid residues. In one embodiment, the third peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 55. In one embodiment, the third peptide linker comprises any of the amino acid sequences as shown in Table 22.

[0061] In one embodiment, fusion of either the VH domain or the VL domain of the second binding site of the first antigen binding molecule with the third Fc region subunit occurs via a fifth peptide linker. In one embodiment, the fifth peptide linker comprises an IgG hinge or a portion or fragment thereof. In one embodiment, the fifth and sixth peptide linkers of the first antigen binding molecule are linked via one or more interchain disulfide bridges. In one embodiment, the fifth and sixth peptide linkers of the first antigen binding molecule comprise one or more cysteine ​​residues that allow for the formation of one or more interchain disulfide bridges between the fifth and sixth peptide linkers. In one embodiment, the fifth and sixth peptide linkers of the first antigen binding molecule comprise one or more cysteine ​​residues that allow for the formation of one or more interchain disulfide bridges between the fifth and sixth peptide linkers, resulting in a disulfide bridge stabilized dimeric peptide linker. In one embodiment, the fifth and sixth peptide linkers comprise an immunoglobulin hinge sequence, preferably derived from an IgG hinge, preferably a human IgG hinge, preferably a fragment thereof, a human IgG1 hinge.

[0062] In one embodiment, the fifth peptide linker has a length of 9 to 29 amino acid residues, preferably 29 amino acid residues. In one embodiment, the fifth peptide linker has a length of 29 amino acid residues. In one embodiment, the fifth peptide linker has a length of 20 amino acid residues. In one embodiment, the sixth peptide linker has a length of 9 amino acid residues. In one embodiment, the fifth peptide linker has a length of 9 to 49 amino acid residues, preferably 29 amino acid residues.

[0063] In one embodiment, the fifth peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:106, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:46. In one embodiment, the fifth peptide linker comprises an amino acid sequence of: AQPAAPAPDAHEAPAPAQGS (SEQ ID NO:31). In one embodiment, the fifth peptide linker comprises an amino acid sequence of: KTHTCPPCP (SEQ ID NO:32). In one embodiment, the fifth peptide linker comprises an amino acid sequence of: AQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO:33).

[0064] In one embodiment, the sixth peptide linker comprises the amino acid sequence of: KTHTCPCP (SEQ ID NO:32). In one embodiment, the sixth peptide linker comprises the amino acid sequence of: DKTHTCPPCP (SEQ ID NO:46). In one embodiment, the fifth peptide linker has the amino acid sequence of: AQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO:33) and the sixth peptide linker has the amino acid sequence of KTHTCPPCP (SEQ ID NO:32). In one embodiment, the fifth peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:54, SEQ ID NO:33, SEQ ID NO:106 and the sixth peptide linker has the amino acid sequence of SEQ ID NO:32.

[0065] In one embodiment of the present disclosure, the second and fourth peptide linkers of the second antigen-binding molecule are linked via two or more interchain disulfide bridges. In one embodiment, the second and fourth peptide linkers of the second antigen-binding molecule contain one or more cysteine ​​residues that allow the formation of one or more interchain disulfide bridges between the second and fourth peptide linkers. In one embodiment, the second and fourth peptide linkers contain one or more cysteine ​​residues that allow the formation of one or more interchain disulfide bridges between the second and fourth peptide linkers, thereby resulting in a disulfide bridge-stabilized dimeric peptide linker. In one embodiment, the second and fourth peptide linkers contain an immunoglobulin hinge sequence, which is preferably derived from an IgG hinge, preferably a human IgG hinge, preferably a fragment thereof, a human IgG1 hinge.

[0066] In one embodiment of the present disclosure, fusion of the second targeting portion of the second antigen-binding molecule with the first Fc region subunit occurs via a peptide linker comprising an IgG hinge or a portion or fragment thereof. In one embodiment, the second peptide linker has a length of 5 to 20 amino acid residues, preferably 15 amino acid residues. In one embodiment, the second peptide linker has a length of 15 amino acid residues. In one embodiment, the second peptide linker comprises the amino acid sequence of the following: EPKSCDKTHTCPPCP (SEQ ID NO: 34). In one embodiment, the second peptide linker comprises the amino acid sequence of DKTHTCPPCP (SEQ ID NO: 46). In one embodiment, the second peptide linker comprises the amino acid sequence of KTHTCPPCP (SEQ ID NO: 32). In one embodiment, the second peptide linker comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 34).

[0067] In one embodiment, the fourth peptide linker has a length of 5 to 20 amino acid residues, preferably 9 to 15 amino acid residues. In one embodiment, the fourth peptide linker has a length of 9 amino acid residues. In one embodiment, the fourth peptide linker has a length of 15 amino acid residues. In one embodiment, the fourth peptide linker comprises the amino acid sequence: KTHTCPPCP (SEQ ID NO: 32). In one embodiment, the fourth peptide linker comprises the amino acid sequence of DKTHTCPPCP (SEQ ID NO: 46). In one embodiment, the fourth peptide linker comprises the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO: 34).

[0068] In one embodiment, the second and fourth peptide linkers have a length of 5 to 20 amino acid residues, preferably 15 amino acid residues. In one embodiment, the second and fourth peptide linkers are identical. In one embodiment, the second and / or fourth peptide comprises an amino acid sequence selected from the group consisting of DKTHTCPPCP (SEQ ID NO: 46), EPKSCDKTHTCPPCP (SEQ ID NO: 34) and KTHTCPPCP (SEQ ID NO: 32).

[0069] In one embodiment, the second peptide linker has a length of 15 amino acid residues and the fourth peptide linker has a length of 9 amino acid residues or 15 amino acid residues. In one embodiment, the second peptide linker has a length of 10 amino acid residues and the fourth peptide linker has a length of 9 amino acid residues or 15 amino acid residues. In one embodiment, the second peptide linker has a length of 5 to 15 amino acid residues and the fourth peptide linker has a length of 9 to 15 amino acid residues.

[0070] In one embodiment, the second peptide linker has the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO:34) and the fourth peptide linker has the amino acid sequence of KTHTCPPCP (SEQ ID NO:32). In one embodiment, the second peptide linker has the amino acid sequence of DKTHTCPPCP (SEQ ID NO:46) and the fourth peptide linker has the amino acid sequence of KTHTCPPCP (SEQ ID NO:32). In one embodiment, the second peptide linker and the fourth peptide linker have the amino acid sequence of EPKSCDKTHTCPPCP (SEQ ID NO:34). In one embodiment, the second peptide linker and the fourth peptide linker have the amino acid sequence of DKTHTCPPCP (SEQ ID NO:34).

[0071] In one embodiment of the present disclosure, the fusion of the complementary VH or VL domain of the second binding site with the first Fc region subunit of the second antigen binding molecule is via a third peptide linker. In one embodiment, the third peptide linker comprises the amino acid sequence: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16).

[0072] In one embodiment of the present disclosure, the first peptide linker has a length of 5 to 45 amino acid residues, the third peptide linker has a length of 5 to 20 amino acid residues, the fifth peptide linker has a length of 9 to 49 amino acid residues, and the second, fourth and sixth peptide linkers each have a length of 5 to 20 amino acid residues.

[0073] In one embodiment of the disclosure, the first, second and / or third binding site comprises an antibody Fv region. In one embodiment, the first, second and / or third binding site is an antibody Fv region. In one embodiment of the disclosure, the second binding site is an antibody Fv region. In one embodiment of the disclosure, the second binding site is an Fv domain.

[0074] In one embodiment of the present disclosure, the distance between the first binding site and the second binding site is 30 Å to 130 Å, preferably 45 Å to 110 Å. In one embodiment, the distance between the first binding site and the second binding site is about 30 Å, about 35 Å, about 40 Å, about 45 Å, about 50 Å, about 55 Å, about 60 Å, about 65 Å, about 70 Å, about 75 Å, about 80 Å, about 90 Å, about 100 Å, about 105 Å, about 110 Å, about 115 Å, about 120 Å, about 125 Å, or about 130 Å.

[0075] In one embodiment, the distance between the first binding site and either the VH or VL domain of the first antigen-binding molecule is 30 Å to 130 Å, preferably 45 Å to 110 Å. In one embodiment, the distance between the first binding site and either the VH or VL domain of the first antigen-binding molecule is about 30 Å, about 35 Å, about 40 Å, about 45 Å, about 50 Å, about 55 Å, about 60 Å, about 65 Å, about 70 Å, about 75 Å, about 80 Å, about 90 Å, about 100 Å, about 105 Å, about 110 Å, about 115 Å, about 120 Å, about 125 Å, or about 130 Å.

[0076] In one embodiment, the distance between the third binding site and the second binding site is 120 Å to 200 Å, preferably 120 Å to 170 Å. In one embodiment, the distance between the first binding site and the second binding site is about 120 Å, about 125 Å, about 130 Å, about 135 Å, about 140 Å, about 145 Å, about 150 Å, about 155 Å, about 160 Å, about 165 Å, about 170 Å, about 175 Å, about 180 Å, about 190 Å, or about 200 Å.

[0077] In one embodiment, the distance between the third binding site of the second antigen-binding molecule and the complementary VH or VL domain is 120 Å to 200 Å, preferably 120 Å to 170 Å. In one embodiment, the distance between the third binding site of the second antigen-binding molecule and the complementary VH or VL domain is about 120 Å, about 125 Å, about 130 Å, about 135 Å, about 140 Å, about 145 Å, about 150 Å, about 155 Å, about 160 Å, about 165 Å, about 170 Å, about 175 Å, about 180 Å, about 190 Å, or about 200 Å.

[0078] In one embodiment, the distance between the first binding site and the second binding site is 30 Å to 130 Å, preferably 45 Å to 110 Å, and the distance between the third binding site and the second binding site is 120 Å to 200 Å, preferably 120 Å to 170 Å. In one embodiment, the distance between the first binding site and either the VH or VL domain of the first antigen-binding molecule is 30 Å to 130 Å, preferably 45 Å to 110 Å, and the distance between the third binding site and the complementary VH or VL domain of the second antigen-binding molecule is 120 Å to 200 Å, preferably 120 Å to 170 Å.

[0079] In one embodiment of the present disclosure, the second antigen is expressed on an immune cell. In one embodiment, the second antigen is a member of a T cell receptor complex. In one embodiment, the member of a T cell receptor complex is CD3. In one embodiment, the second antigen is human CD3. In one embodiment, the second antigen is human CD3ε. In one embodiment, the second antigen is a polypeptide comprising the amino acid sequence of SEQ ID NO:57.

[0080] In one embodiment of the present disclosure, the second binding site is an antibody Fv region. In one embodiment of the present disclosure, the second binding site is an antibody Fv region with specificity for CD3. In one embodiment, the second binding site is specific for CD3. In one embodiment, the second binding site is specific for human CD3ε.

[0081] In an embodiment of the disclosure, the VH domain of the second binding site specific for CD3 comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 59. In one embodiment of the disclosure, the VL domain of the second binding site specific for CD3 comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 60.

[0082] In one embodiment, (i) the VH domain of the second binding site of the first antigen binding molecule comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:59, and the complementary VL domain of the second binding site of the second antigen binding molecule comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:60, or (ii) the VL domain of the second binding site of the first antigen binding molecule comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:60, and the complementary VH domain of the second binding site of the second antigen binding molecule comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:59.

[0083] In one embodiment, the VH domain of the second binding site specific for CD3 comprises the amino acid sequence of SEQ ID NO: 1 and the VL domain of the second binding site specific for CD3 comprises the amino acid sequence of SEQ ID NO: 2, or the VH domain of the second binding site specific for CD3 comprises the amino acid sequence of SEQ ID NO: 59 and the VL domain of the second binding site specific for CD3 comprises the amino acid sequence of SEQ ID NO: 60. In one embodiment, the second binding site specific for CD3 has a VH domain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 59 and a VL domain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 60.

[0084] In one embodiment, the second binding site specific for CD3 comprises (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 1 and a VL domain comprising the amino acid sequence of SEQ ID NO: 2, or (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 59 and a VL domain comprising the amino acid sequence of SEQ ID NO: 60. In one embodiment, the Fv region specific for CD3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 59 and a VL domain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 60. In one embodiment, the Fv region specific for CD3 comprises (i) a VH domain comprising the amino acid sequence of SEQ ID NO: 1 and a VL domain comprising the amino acid sequence of SEQ ID NO: 2, or (ii) a VH domain comprising the amino acid sequence of SEQ ID NO: 59 and a VL domain comprising the amino acid sequence of SEQ ID NO: 60.

[0085] In one embodiment, the second binding site specific for CD3 comprises (i) a VH domain having a VH domain consisting of an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 4, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 5, or (ii) a VH domain having an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 61, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 62, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 5. In one embodiment, the second binding site specific for CD3 comprises (i) a VL domain having an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 8, or (ii) a VL domain having an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 63, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 8.

[0086] In one embodiment, the second binding site specific for CD3 comprises: a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO:3; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO:4; c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO:5; d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO:6; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7; and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO:8 and a VH domain having the following structure:

[0087] In one embodiment, the second binding site specific for CD3 comprises: a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 61; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 62 c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO:5 d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 63 e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7; and f) an LCDR3 region comprising the amino acid sequence of SEQ ID NO:8 and a VH domain having the following structure:

[0088] In one embodiment, the second binding site specific for CD3 comprises: (i) a VH domain having an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 4, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 5, and a VL having an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 8, or (ii) a VH domain having an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 61, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 62, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 5, and a VL domain comprising an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 63, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 region having the amino acid sequence of SEQ ID NO: 8.

[0089] In one embodiment, the Fv region specific for CD3 comprises: (i) a VH domain having an HCDR1 region comprising the amino acid sequence of SEQ ID NO:3, an HCDR2 region comprising the amino acid sequence of SEQ ID NO:4, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO:5, and a VL domain having an LCDR1 region comprising the amino acid sequence of SEQ ID NO:6, an LCDR2 region comprising the amino acid sequence of SEQ ID NO:7, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO:8, or (ii) a VH domain having an HCDR1 region comprising the amino acid sequence of SEQ ID NO:61, an HCDR2 region comprising the amino acid sequence of SEQ ID NO:62, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO:5, and a VL domain comprising an LCDR1 region comprising the amino acid sequence of SEQ ID NO:63, an LCDR2 region comprising the amino acid sequence of SEQ ID NO:7, and an LCDR3 region having the amino acid sequence of SEQ ID NO:8.

[0090] In one embodiment of the present disclosure, the first antigen binding molecule comprises a VH domain of an antibody Fv region specific for CD3 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 59. In one embodiment of the present disclosure, the first antigen binding molecule comprises a VL domain of an antibody Fv region specific for CD3 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 60. In one embodiment of the present disclosure, the second antigen binding molecule comprises a VH domain of an antibody Fv region specific for CD3 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 59. In one embodiment of the present disclosure, the second antigen binding molecule comprises a VL domain of an antibody Fv region specific for CD3 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 60.

[0091] In one embodiment of the present disclosure, (i) the first antigen binding molecule comprises a VH domain of an antibody Fv region specific for CD3 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 59, and the second antigen binding molecule comprises a complementary VL domain of an antibody Fv region specific for CD3 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 60, or (ii) the first antigen binding molecule comprises a VL domain of an antibody Fv region specific for CD3 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 60, and the second antigen binding molecule comprises a complementary VH domain of an antibody Fv region specific for CD3 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 59.

[0092] In one embodiment of the present disclosure, the first antigen and the third antigen are present on the same cell. In one embodiment, the cell is a tumor cell. In one embodiment, the first antigen and the third antigen are tumor associated antigens. In one embodiment, the first antigen and the third antigen are present on the same cell and the second antigen is present on different cells. In one embodiment, the first antigen and the third antigen are present on a tumor cell and the second antigen is present on an immune cell. In one embodiment, the second antigen is present on a T cell. In one embodiment, the second antigen is CD3. In one embodiment, the second antigen is CD3ε. In one embodiment, the first antigen and the second antigen are the same. In one embodiment, the first antigen and the third antigen are the same. In one embodiment, the first antigen and the second antigen are different. In one embodiment, the first antigen, the second antigen, and the third antigen are different.

[0093] In one embodiment of the present disclosure, the first antigen comprises a first antigen epitope. In one embodiment, the second antigen comprises a second antigen epitope. In one embodiment, the third antigen comprises a third antigen epitope. In one embodiment of the present disclosure, the first antigen epitope and the second antigen epitope are identical. In one embodiment of the present disclosure, the first antigen epitope and the third antigen epitope are identical. In one embodiment of the present disclosure, the first antigen epitope and the second antigen epitope are different. In one embodiment of the present disclosure, the first antigen epitope and the third antigen epitope are different. In one embodiment of the present disclosure, the first antigen epitope, the second antigen epitope, and the third antigen epitope are different.

[0094] In one embodiment, the first antigen epitope is closer to the cell surface than the second antigen epitope. In one embodiment, the first antigen epitope is closer to the cell surface than the third antigen epitope. In one embodiment, the second antigen epitope is closer to the cell surface than the first antigen epitope. In one embodiment, the third antigen epitope is closer to the cell surface than the first antigen epitope. In one embodiment, the first antigen epitope is at least 15 Å closer to the cell surface than the second antigen epitope. In one embodiment, the first antigen epitope is at least 15 Å closer to the cell surface than the third antigen epitope. In one embodiment, the second antigen epitope is at least 15 Å closer to the cell surface than the first antigen epitope. In one embodiment, the third antigen epitope is at least 15 Å closer to the cell surface than the first antigen epitope.

[0095] In one embodiment, the first and second antigen epitopes are at equal distances to the cell surface. In one embodiment, the first and third antigen epitopes are at equal distances to the cell surface. In one embodiment, the distance from the first antigen epitope to the cell surface is shorter than the distance from the second antigen epitope to the cell surface. In one embodiment, the distance from the first antigen epitope to the cell surface is shorter than the distance from the third antigen epitope to the cell surface. In one embodiment, the distance from the second antigen epitope to the cell surface is shorter than the distance from the first antigen epitope to the cell surface. In one embodiment, the distance from the third antigen epitope to the cell surface is shorter than the distance from the first antigen epitope to the cell surface.

[0096] In one embodiment of the present disclosure, the first targeting portion of the first antigen binding molecule binds to an antigen epitope closer to the cell surface than the antigen epitope bound by the second targeting portion of the second antigen binding molecule. In one embodiment, the first targeting portion of the first antigen binding molecule binds to an antigen epitope at least 15 Å closer to the cell surface than the antigen epitope bound by the second targeting portion of the second antigen binding molecule. In one embodiment of the present disclosure, the first targeting portion of the first antigen binding molecule binds to a membrane-proximal antigen epitope on the cell surface, and the second antigen binding molecule binds to a membrane-distal antigen epitope on the cell surface.

[0097] In one embodiment, the second targeting portion of the second antigen binding molecule binds to a third antigen epitope that is closer to the cell surface than the first antigen epitope that the first targeting portion of the first antigen binding molecule binds to. In one embodiment, the second targeting portion of the second antigen binding molecule binds to a third antigen epitope that is at least 15 Å closer to the cell surface than the first antigen epitope that the first targeting portion of the first antigen binding molecule binds to. In one embodiment of the present disclosure, the first targeting portion of the first antigen binding molecule binds to a membrane distal antigen epitope on the cell surface, and the second antigen binding molecule binds to a membrane proximal antigen epitope on the cell surface.

[0098] In one embodiment of the present disclosure, the first and / or second Fc region comprises one or more amino acid modifications that promote binding of the first and second Fc region subunits and the third and fourth Fc region subunits. In one embodiment of the present disclosure, the CH3 domain of each Fc domain subunit comprises an amino acid modification that promotes binding of the first and second Fc region subunits and the third and fourth Fc region subunits. In one embodiment of the present disclosure, each CH3 domain of the first and second Fc domain subunits and each CH3 domain of the third and fourth Fc domain subunits comprises an amino acid modification that promotes binding of the first and second Fc region subunits and the third and fourth Fc region subunits, respectively. In one embodiment, the CH3 domain of the first and second Fc domain subunits and the CH3 domain of the third and fourth Fc domain subunits comprises an amino acid modification that promotes binding of the first and second Fc region subunits and the third and fourth Fc region subunits, respectively.

[0099] In one embodiment of the disclosure, a first and second Fc region subunit are provided that comprise one or more amino acid modifications that facilitate binding of the first and second Fc region subunits. In one embodiment of the disclosure, a third and fourth Fc region subunit are provided that comprise one or more amino acid modifications that facilitate binding of the third and fourth Fc region subunits.

[0100] In one embodiment, in the CH3 domain of the first and / or third Fc region subunit, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), the serine residue at position 354 is replaced with a cysteine ​​residue (S354C), and in the CH3 domain of the second and / or fourth Fc region subunit, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), the threonine residue at position 366 is replaced with a serine residue (T366S), the leucine residue at position 368 is replaced with an alanine residue (L368A), and the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C), using the numbering according to the EU index.

[0101] In one embodiment of the disclosure, the first and / or second Fc region is engineered to have altered binding affinity for an Fc receptor and / or C1q and / or altered effector function compared to the wild-type Fc region. In one embodiment, the first and / or second Fc region has increased binding affinity for an Fc receptor and / or C1q and / or increased effector function compared to the wild-type Fc region. In one embodiment, the first and / or second Fc region has decreased binding affinity for an Fc receptor and / or C1q and / or decreased effector function compared to the wild-type Fc region. In one embodiment, the first and / or second Fc region has substantially no binding affinity for an Fc receptor and / or C1q and / or substantially no effector function compared to the wild-type Fc region. In one embodiment, the first and / or second Fc region has no binding affinity for an Fc receptor and / or C1q and / or no effector function compared to the wild-type Fc region.

[0102] In one embodiment of the disclosure, the first and second and / or third and fourth Fc region subunits are mutated in at least one of five amino acid residues at positions corresponding to positions L234, L235, G237, A330, P331, numbering according to the EU index for human IgG1. In one embodiment, the first and second and / or third and fourth Fc region subunits are mutated in at least one of five amino acid residues at positions corresponding to positions L234, L235, G237, A330, P331, numbering according to the EU index for human IgG1, such that the first and / or second Fc region has substantially no binding affinity for an Fc receptor and / or C1q and / or substantially no effector function compared to a wild-type human IgG1 Fc region. In one embodiment, in the first and second and / or third and fourth Fc region subunits, at least one of the five amino acid residues at positions corresponding to L234, L235, G237, A330, P331, numbering according to the EU index for human IgG1, is mutated to A, E, A, S, and S, respectively. In one embodiment, in the first and second and / or third and fourth Fc region subunits, at least one of the five amino acid residues at positions corresponding to L234, L235, G237, A330, P331, numbering according to the EU index for human IgG1, is mutated to A, E, A, S, and S, respectively, wherein the first and / or second Fc region has substantially no binding affinity for an Fc receptor and / or C1q and / or substantially no effector function compared to a wild-type Fc region. In one embodiment, in the first and second and / or third and fourth Fc region subunits, at least five amino acid residues at positions corresponding to L234, L235, G237, A330 and P331, numbered according to the EU index for human IgG1, are mutated to A, E, A, S and S, respectively.In one embodiment, in the first and second and / or third and fourth Fc region subunits, at least five amino acid residues at positions corresponding to L234, L235, G237, A330, P331, numbered according to the EU index for human IgG1, are mutated to A, E, A, S, and S, respectively, such that the engineered Fc region has substantially no binding affinity for an Fc receptor and / or C1q and / or substantially no effector function compared to a wild-type Fc region.

[0103] In one embodiment, the present disclosure provides a set of antigen-binding molecules consisting of or comprising a first and a second antigen-binding molecule according to the present disclosure for use as a medicament. In one embodiment, the present disclosure provides a multispecific antibody consisting of a set of antigen-binding molecules according to the present disclosure. In one embodiment, the present disclosure provides a kit comprising a set of antigen-binding molecules consisting of or comprising a first and a second antigen-binding molecule according to the present disclosure. In one embodiment, the present disclosure provides a first pharmaceutical composition comprising a first antigen-binding molecule according to the present disclosure and a second pharmaceutical composition comprising a second antigen-binding molecule according to the present disclosure. In one embodiment, the present disclosure provides a kit comprising a first and a second pharmaceutical composition according to the present disclosure.

[0104] In one embodiment, the antigen-binding molecules included in the set of antigen-binding molecules according to the present disclosure are isolated antigen-binding molecules. In one embodiment, the antigen-binding molecules included in the set of antigen-binding molecules according to the present disclosure are recombinant antigen-binding molecules. In one embodiment, the antigen-binding molecules are isolated recombinant antigen-binding molecules.

[0105] In one embodiment, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antigen-binding molecule according to the present disclosure. In one embodiment, an antigen-binding molecule according to the present disclosure is encoded by a nucleic acid composition of the present disclosure. In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition encoding an antigen-binding molecule of the present disclosure. In one embodiment, the present disclosure provides a host cell comprising a vector composition of the present disclosure or a nucleic acid composition encoding an antigen-binding molecule of the present disclosure. In one embodiment, the present disclosure provides a host cell comprising a nucleic acid composition of the present disclosure or a vector composition of the present disclosure encoding an antigen-binding molecule of the present disclosure. In one embodiment, the present disclosure provides a host cell, wherein the host cell is a eukaryotic cell, particularly a mammalian cell. [Brief description of the drawings]

[0106] [Figure 1] 1 shows the design of a (1 1 / 2) antigen-binding molecule according to the present disclosure. [Figure 1A] 1 shows the structure of the (1 1 / 2)B027 antigen binding molecule according to the present disclosure. This structure includes an N-terminal Fab as a targeting moiety. The C-terminal 1 / 2 Fv domain is provided by either an unpaired VH or VL antibody domain (FIG. 1A depicts an unpaired VL domain). The unpaired variable domain is fused to the C-terminus of the Fab heavy chain, for example, via a short peptide linker. [Figure 1B]1 shows the structure of the B036 (Fc-KiH) 1 1 / 2 antigen binding molecule according to the present disclosure. The basic structure of the B036 format is based on the B027 format, but differs in the addition of a dimeric Fc region incorporated at its C-terminus as a half-life extension moiety. The structure of the B036 format includes an N-terminal Fab as a targeting moiety followed by either an unpaired VH or VL domain of an antibody Fv domain (FIG. 1B depicts an unpaired VL domain). The unpaired variable domain is fused at its N-terminus to the C-terminus of the Fab heavy chain, e.g., via a short peptide linker, and fused at its C-terminus to the N-terminus of one of the two Fc region subunits. The two peptide linkers present at the N-terminus of each Fc region subunit may contain interchain cysteines that allow for the formation of stabilizing disulfide bridges. Heterodimerization of the polypeptide chains that form the two Fc region subunits is promoted by modification of the CH3 domain of each Fc region subunit by means of disulfide stabilization and knob-into-holes technology (KiH). [Figure 1C] 1 shows the structure of the (1 1 / 2)B038 (Fc-KiH) antigen binding molecule according to the present disclosure. The B038 format provides an alternative embodiment to the B036 format. In this case, the entire Fc region is incorporated between the N-terminal Fab and the unpaired VH or VL domain of the antibody Fv domain (FIG. 1C depicts the unpaired VL domain). The entire Fc region serves as a spacer between the Fab and 1 / 2 Fv domains and also enhances the serum stability of the molecule. In this format, the Fab heavy chain is fused at its C-terminus to the N-terminus of one of the two Fc region subunits carrying knob mutations, which is fused at its C-terminus to the unpaired VH or VL domain. The two peptide linkers present at the N-terminus of each Fc region subunit contain interchain cysteines, allowing the formation of stabilizing disulfide bridges between the two linkers. Heterodimerization of the two polypeptide chains forming the two Fc region subunits is promoted by modification of the CH3 domain of each Fc region subunit by means of disulfide stabilization and knobs-into-holes technology (KiH). [Diagram 2]Various exemplary possibilities for combining antigen-binding molecules of the B027, B036 and B038 formats of the present disclosure are shown. To provide a functional combination of two antigen-binding molecules, one antigen-binding molecule must carry an unpaired VH domain of an antibody Fv domain, and the second antigen-binding molecule must carry a complementary unpaired VL domain of the same antibody Fv domain. The non-covalent binding of the VH and VL domains achieves the functional complementarity and formation of the antibody Fv domain when the two antigen-binding molecules bind to their target antigens via the targeting moiety. [Figure 2A] The combination of two antigen-binding molecules in the B027 format without half-life extension moieties is shown. This combination results in the on-cell formation of a trispecific antibody that appears to be symmetric. Thus, the distance between each of the two Fab targeting moieties and the newly formed antibody Fv domain (such as the CD3-specific Fv domain) is nearly identical. Each of the two Fabs can target a different antigen or antigen epitope on the same target cell. CD3 binding of the newly formed antibody Fv domain can redirect T cells to the target cell. The small size of the two antigen-binding molecules and the close distance between the targeting domain and the newly formed CD3-specific Fv domain allow the formation of a narrow immune synapse between the target cell and the cytotoxic T cell, which is a prerequisite for optimal target cell killing. In addition, targeting of membrane-proximal epitopes can be advantageous for small size molecules compared to large and bulky antibodies such as conventional IgG format-based antibody formats. [Figure 2B] Each represents a combination of two antigen-binding molecules of the B036 format incorporating a half-life extending moiety. This combination results in the on-cell formation of a trispecific antibody that appears to be symmetric. Thus, the distance between each of the two Fab targeting moieties and the newly formed antibody Fv domain (such as the CD3-specific Fv domain) is nearly identical. This combination combines the advantages of the minimal B027 format in terms of the short distance between the targeting domain and the CD3 Fv binding domain, and the extended serum stability due to the presence of two full Fc regions. [Figure 2C] Each represents a combination of two antigen-binding molecules in B038 incorporating a half-life extending moiety. This combination also results in on-cell formation of a trispecific antibody that appears to be symmetric. Thus, the distance between each of the two Fab targeting moieties and the newly formed antibody Fv domain (such as the CD3-specific Fv domain) is nearly identical. This combination also results in extended serum stability due to the presence of the entire Fc region in each of the two antigen-binding molecules. However, compared to the combinations of B027 or B036, the distance between the Fab and the newly formed (CD3-specific) Fv domain is greater, resulting in the formation of a more extensive immune synapse between the target cell and the cytotoxic T cells. [Figure 2D] The combination of one antigen-binding molecule in the B027 format and one antigen-binding molecule in the B036 format is shown. This combination results in a trispecific antibody that is nearly symmetrical in appearance, although the two molecules are not identical. However, the distance between each of the two Fab targeting moieties and the newly formed antibody Fv domain (such as the CD3-specific Fv domain) is within the same range (Figure 2D depicts an unpaired VL domain in the B036 format and an unpaired VH domain in the B027 format. However, the two domains could be swapped within the two formats). Since only one full IgG Fc region is present in the on-cell formed trispecific antibody, the antibody appears similar in shape to a conventional IgG molecule. The combination of B027 and B036 formats extends the serum stability of the antigen-binding molecule in the B036 format. In contrast, the molecules in the B027 format are cleared from the blood relatively quickly. This combination also allows for a shorter distance between the Fab targeting moiety and the newly formed (CD3-specific) Fv domain, thus resulting in the formation of a narrow immune synapse between the target cell and the immune cell. [Figure 2E]The combination of one antigen-binding molecule in the B027 format and one antigen-binding molecule in the B038 format is shown. This combination results in an on-cell formation of a trispecific antibody that appears to be asymmetric. In this regard, the distance between each of the two Fab targeting moieties and the newly formed antibody Fv domain (such as the CD3-specific Fv domain) is quite different. This specific combination allows optimal targeting of epitopes with different distances to the cell surface (Figure 2E depicts the unpaired VL domain of an antigen-binding molecule in the B038 format and the unpaired VH domain of an antigen-binding molecule in the B027 format. However, the two domains may be exchanged within the two formats). The Fc-bearing antigen-binding molecule in the B038 format is preferably selected to target a more membrane-proximal epitope, whereas the Fc-free antigen-binding molecule in the B027 format is preferably selected to target a more membrane-distal epitope on the target cell. In this setting, the Fc portion of the B038 format acts as an extension or stalk to bring its unpaired variable domain into close proximity with the complementary variable domain of an antigen binding molecule of the B027 format. [Figure 2F]The combination of one antigen-binding molecule in the B036 format and one antigen-binding molecule in the B038 format is shown (Figure 2F depicts the unpaired VL domain of the antigen-binding molecule in the B036 format and the unpaired VH domain of the antigen-binding molecule in the B038 format). However, the two domains may also be swapped within the two formats). This combination results in an on-cell formation of a trispecific antibody that appears to be asymmetric. Thus, the distance between each of the two Fab targeting sites and the newly formed antibody Fv domain (such as the CD3-specific Fv domain) is not the same, but rather quite different. This combination also allows optimal targeting of two different epitopes with different distances to the cell surface. In this setting, the antigen-binding molecule in the B038 format is selected to target a more membrane-proximal epitope, whereas the antigen-binding molecule in the B036 format is selected to target a more membrane-distal epitope on the target cell. The Fc portion of the B038 format acts as an extension or stalk to bring its unpaired variable domain into close proximity with the complementary variable domain of an antigen binding molecule of the B036 format. [Diagram 3] Right panel: Crystal structure of the extracellular domain of epidermal growth factor receptor in complex with the Fab fragment of cetuximab (Erbitux). This structure was obtained from the Protein Data Bank under PDB ID 1YY9. This structure is oriented in its relative position to the cell membrane. The dotted circle indicates the membrane distal binding region / epitope of cetuximab on EGFR. Left panel: Crystal structure of the extracellular domain of HER2 in complex with the Fab fragment of trastuzumab (Herceptin). This structure was obtained from the Protein Data Bank under PDB ID 1N8Z. This structure is oriented in its relative position to the cell membrane. The dotted circle indicates the membrane proximal binding region / epitope of trastuzumab on HER2. [Figure 4]1 shows the structure of the (2 1 / 2)B064 (Fc-KiH) antigen binding molecule according to the present disclosure, as well as various exemplary possibilities for combining antigen binding molecules of the B064 format with antigen binding molecules of the B064, B027 and B036 formats. [Figure 4A] 1 shows the structure of the (2 1 / 2)B064 (Fc-KiH) antigen binding molecule according to the present disclosure. The B064 format provides an alternative embodiment to the B038 format, differing only in bivalent tumor targeting. Thus, a second Fab (identical to the first Fab fused to the first Fc region subunit, but with a knob mutation in its CH domain) is fused to the N-terminus of a second Fc region subunit with a hole mutation. [Figure 4B-4D] Various possibilities are shown for combining antigen-binding molecules in the B064 format with antigen-binding molecules in the B064 format (FIG. 4B), B027 format (FIG. 4C) or B036 format (FIG. 4D). Unpaired antibody variable domains may also be exchanged between the antigen-binding molecules. As with the B038 format, combining the B064 format with the B027 format or the B036 format results in on-cell formation of a trispecific antibody that appears asymmetric. This particular combination also allows optimal targeting of epitopes with different distances to the cell surface, for example, in this case the antigen-binding molecules in the B064 format are selected to target more membrane-proximal epitopes and the antigen-binding molecules in the B027 or B036 format are selected to target more membrane-distal epitopes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] definition The term "antigen" or "target antigen" as used herein refers to any molecule of interest that can bind to one of the binding sites present in the antigen-binding molecule of the present disclosure. Typically, the antigen is a peptide, protein or other proteinaceous molecule. Alternatively, the antigen may be other organic or inorganic molecules, such as carbohydrates, fatty acids, lipids, dyes or fluorescent dyes.

[0108] The term "polypeptide" as used herein refers to a polymer of amino acid residues and does not refer to a specific length of the product. The term applies to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular amino acid sequence of a polypeptide also implicitly encompasses conservatively modified variants thereof (e.g., by replacing an amino acid residue with another amino acid residue having similar structural and / or chemical properties). A polypeptide may be derived from a natural organism or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. A polypeptide may be produced in any manner, including chemical synthesis.

[0109] The term "antigen-binding molecule" as used herein broadly refers to a proteinaceous molecule that specifically binds to at least one antigen. An antigen-binding molecule may be composed of one or more polypeptides. Examples of antigen-binding molecules are immunoglobulins and their derivatives and / or fragments. An antigen-binding molecule according to the present disclosure may be based on a conventional immunoglobulin (e.g., IgG), in particular composed of a half IgG molecule. An antigen-binding molecule as disclosed herein is composed of at least one targeting moiety (such as an antibody Fab fragment) and either an additional VH or VL domain of an antibody Fv domain, where neither the VH nor the VL domain can bind to its antigen by itself. Thus, an antigen-binding molecule according to the present disclosure may also be designated as a 1+1 / 2 or 1 1 / 2 antigen or 2+1 / 2 or 2 1 / 2 antigen-binding molecule, since it incorporates a half Fv domain (1 / 2 Fv domain) or half a binding site and one or two Fv domains or the entire binding site.

[0110] As used herein, the term "targeting moiety" refers to any polypeptide or protein capable of specifically binding to an antigen. Non-limiting examples of targeting moieties that can be used in antigen-binding molecules according to the present disclosure include antibodies or antibody fragments, cytokines, or ligands for receptors.

[0111] As used herein, the term "binding site" or "antigen-binding site" refers to a structure formed by a protein that can bind or specifically bind to an antigen. A binding site does not necessarily have to be a series of contiguous amino acids, but may be amino acids in a single polypeptide chain. For example, in an Fv produced from two different polypeptide chains, the binding site is composed of a series of amino acids in the VL and VH that interact with the antigen, but generally not always in one or more CDRs of each variable region. In certain embodiments, the binding site is, comprises, or is formed by a complementary antibody variable heavy chain (VH) and light chain (VL) pair. The VH and VL that form the binding site may be present in either a single polypeptide chain or different polypeptide chains. In a preferred embodiment, the binding site is, comprises, or is formed by a VH present on a first antigen-binding molecule according to the present disclosure and a complementary VL present on a second antigen-binding molecule according to the present disclosure, or vice versa. In some embodiments, the binding site has one VH and one VL. In certain embodiments, the binding site consists of one or more CDRs of an antibody, hi other embodiments, the binding site is derived from an antibody mimetic, such as, for example, an affibody molecule, an alphabody, an anticalin, an avimer, a DARPin, a finomer, a Kunitz domain peptide, a helix-turn-helix peptide, or a monobody.

[0112] The term "antibody" molecule or "immunoglobulin" (Ig) molecule, as used herein, refers to a protein that contains at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds and that interacts with an antigen. Each heavy chain (HC) is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain (LC) is composed of a light chain variable domain (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL domains are further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with highly conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable domains of the heavy and light chains (VH and VL) comprise or form a "binding site" or "antigen-binding site" that selectively interacts with or binds to an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies can be of 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 of structural and functional homology.

[0113] The term "antibody fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, stabilization of spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, a monovalent fragment consisting of VL, VH, CL and CH1 domains, where the Fab heavy chain (HC) is formed by the VH and CH1 domains (VH-CH1) and the Fab light chain is formed by complementary VL and CL domains (VL-CL). Thus, the Fab heavy and Fab light chains are complementary to each other; F(ab)2, a bivalent fragment comprising two Fabs linked by disulfide bridges at the hinge region; Fd fragment consisting of the VH and CH1 domains; Fv fragment or Fv region or Fv domain consisting of a dimer of one VL and one VH domain. Thus, the VH and VL domains of an Fv fragment or Fv region are complementary to each other; a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and isolated complementarity determining regions (CDRs). Furthermore, although the two variable domains of an Fv fragment or Fv region are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows the domains to be produced as a single protein chain (referred to herein as a "single-chain Fv" or "scFv"; e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883) in which the VL and VH regions pair to form a monovalent molecule. Such single chain antibodies are intended to be encompassed by the term “antibody fragment.” Antibody fragments are obtained using conventional techniques known to those of skill in the art, and screened for utility in the same manner as are intact antibodies.Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136). Antibody fragments can be grafted into scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments can be assembled into single-chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641,870).

[0114] The term immunoglobulin (Ig) "hinge" as used herein refers to one of the two polypeptides that form the dimeric "hinge region" of an immunoglobulin. The hinge comprises the portion of the immunoglobulin heavy chain that connects the CH1 domain to the CH2 domain. Thus, a naturally occurring immunoglobulin is composed of two identical hinges that are linked through one or more disulfide bridges formed by the interchain cysteines present in the two hinges. In other words, a naturally occurring immunoglobulin is composed of a dimeric disulfide-stabilized hinge region that connects the two Fab arms of the immunoglobulin to the Fc region. The hinge can be further divided into three different domains: the upper hinge, the middle hinge, and the lower hinge (Roux et al., J. Immunol. 1998 161:4083).

[0115] The term "Fc region" as used herein refers to two Fc region subunits that can stably associate with each other, thereby forming a dimeric C-terminal region of an immunoglobulin. Thus, the two Fc region subunits are complementary to each other. The Fc region of a normal IgG molecule or an antigen-binding molecule according to the present disclosure exists as a dimer, each subunit of which contains CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc region are capable of stable binding with each other.

[0116] As used herein, "Fc region subunit" refers to one of two polypeptides that form the dimeric Fc region of an immunoglobulin or antigen-binding molecule according to the present disclosure. (i.e., a polypeptide that includes the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association). Thus, the two Fc region subunits that form the dimeric Fc region are complementary to each other. For example, an IgG Fc region subunit includes an IgG CH2 and an IgG CH3 constant domain. The term encompasses native or wild-type sequence Fc region subunits and mutant or modified Fc region subunits. Although the boundaries of the Fc region subunits of an IgG heavy chain may vary slightly, human IgG heavy chain Fc region subunits are usually defined as extending from Cys226, or from Pro230, to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region subunit may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0117] The term "multispecific" means that an antibody or antigen-binding molecule can specifically bind to two or more different antigens. Generally, a multispecific antibody or antigen-binding molecule contains two or more antigen-binding sites, each specific for a different antigen or epitope. The term "bispecific" means that an antibody or antigen-binding molecule can specifically bind to two different antigens. Usually, a bispecific antigen-binding molecule has two antigen-binding sites, each specific for a different antigen or epitope.

[0118] As used herein, the terms "specifically bind to," "specifically bind to," "specific for / for," or "specifically recognize," and the like, refer to a measurable and reproducible interaction, such as the binding of a target antigen with an antibody, antibody fragment, or antigen-binding molecule disclosed herein, which determines the presence of the target antigen in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody, antibody fragment, or antigen-binding molecule disclosed herein that specifically binds to a target antigen (which may be an antigen or an epitope of an antigen) is an antibody, antibody fragment, or antigen-binding molecule that binds to this target with higher affinity, avidity, more readily, and / or with longer duration than it binds to other target antigens. In certain embodiments, the antibody, antibody fragment, or antigen-binding molecule specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but is not required to include, exclusive binding. The antibody, antibody fragment, or antigen-binding molecule disclosed herein specifically binds to an antigen. Methods for determining whether two molecules specifically bind are known in the art, and include, for example, standard ELISA assays. Scoring can be performed by standard colorimetric phenomena (e.g. secondary antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide). Reactions in specific wells are scored by optical density, e.g. at 450 nm. A typical background (=negative reaction) may be 0.1 OD; a typical positive reaction may be 1 OD. This means that the positive / negative difference may be more than 5-fold. In general, the determination of binding specificity is usually performed not with a single reference antigen, but with a set of 3-5 unrelated antigens, such as milk powder, BSA, transferrin, etc.

[0119] The term "epitope" or "antigenic epitope" refers to a site on a polypeptide or protein (e.g., a contiguous stretch of amino acids or a conformational arrangement of distinct regions of non-contiguous amino acid residues) that is specifically recognized by an antibody, antibody fragment, or antigen-binding molecule disclosed herein, or a T-cell receptor, or that otherwise interacts with a molecule. In general, an epitope is a chemically active surface grouping of molecules, such as amino acids or carbohydrate or sugar side chains, and generally may have specific three-dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by those skilled in the art, an epitope can be virtually anything to which an antibody or antigen-binding molecule can specifically bind. An epitope can include residues to which an antibody or antigen-binding molecule binds, and can be either "linear" or "conformational". A "linear epitope" refers to an epitope in which all of the points of interaction between the protein and the interacting molecule (such as an antibody) are linear (contiguous) along the primary amino acid sequence of the protein. A "conformational epitope" refers to an epitope in which non-contiguous amino acid residues are assembled in a three-dimensional spatial arrangement. In the case of a conformational epitope, the points of interaction are present across the amino acid residues on the protein that are separated from each other. For example, an epitope can be one or more amino acid residues within a stretch of amino acid residues as shown by peptide mapping or HDX, or one or more individual amino acid residues as shown by X-ray crystallography. "Binds to the same epitope" refers to the ability of an antibody, antibody fragment, or antigen-binding molecule to bind to a particular antigen and bind to the same epitope as an exemplary antibody or antigen-binding molecule when the same epitope mapping technique is used to compare the antibodies or antigen-binding molecules. The epitopes of the exemplary antibodies, antigen-binding molecules, and other antibodies and antigen-binding molecules can be determined using epitope mapping techniques. Epitope mapping techniques are well known in the art. For example, conformational epitopes are readily identified by determining the spatial arrangement of amino acids, for example, by hydrogen / deuterium exchange, X-ray crystallography, and two-dimensional nuclear magnetic resonance.

[0120] The terms "engineered" or "modified" as used herein include the manipulation of nucleic acids or polypeptides by synthetic means (e.g., recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or some combination of these techniques). Preferably, an antibody, antibody fragment or antigen-binding molecule according to the present disclosure is engineered or modified to improve one or more properties, such as antigen binding, stability, half-life, effector functions, immunogenicity, safety, etc.

[0121] The term "valency" as used herein refers to the presence of a defined number of antigen-binding sites in an antigen-binding molecule.

[0122] As used herein, the terms "first," "second," "third," "fourth," "fifth," and "sixth" and "seventh," with respect to Fab and / or Fv regions, Fc regions, Fc region subunits, peptide linkers, spacers, or polypeptides, etc., are used to distinguish when more than one of each type of component is present. The use of these terms is not intended to confer a particular order or orientation within the antigen-binding molecules, unless explicitly stated.

[0123] A "modification that promotes binding of the first and second Fc region subunits" refers to a manipulation of the polypeptide backbone or post-translational modification of the Fc region subunit that reduces or prevents binding of a polypeptide comprising the Fc region subunit to the same polypeptide to form a homodimer. Modifications that promote binding as used herein include, in particular, individual modifications made to each of the two Fc region subunits (i.e., the first and second Fc region subunits) that are desired to be bound, where the modifications are complementary to each other to promote binding of the two Fc region subunits. For example, a modification that promotes binding can alter the structure or charge of one or both of the Fc region subunits to make their binding sterically or electrostatically favorable, respectively. Thus, although heterodimerization occurs between a polypeptide comprising a first Fc region subunit and a polypeptide comprising a second Fc region subunit, they are not considered to be identical in the sense that the separate components (e.g., Fab, Fv) fused to each of the subunits are not the same.

[0124] As used herein, an "amino acid residue" or "amino acid" is designated either by its full name or by the standard three letter or one letter amino acid code. By "naturally occurring amino acid" is meant the following amino acids:

[0125] [Table 1]

[0126] The term "amino acid mutation" as used herein is meant to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be made, so long as the final construct has the desired properties, such as reduced binding to Fc receptors or increased binding to another peptide. Deletions and insertions in amino acid sequences include deletions and insertions at the amino and / or carboxy termini of amino acid residues. Particular amino acid mutations are amino acid substitutions. Amino acid substitutions include substitutions with non-naturally occurring amino acids or substitutions with naturally occurring amino acid derivatives of the 20 standard amino acids. Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods include site-directed mutagenesis, PCR, gene synthesis, and the like. Methods other than genetic engineering, such as methods to modify the side chain groups of amino acid residues by chemical modification, are also believed to be useful. Various designations may be used herein to refer to the same amino acid mutation. For example, a glyince to alanine substitution at position 327 in the Fc region can be represented as 237A, G337, G337A, or Gly329Ala.

[0127] The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0128] As used herein, "treatment", "treat" or "treating" refers to a clinical intervention that seeks to alter the natural course of a disease in the individual being treated, and can be performed for prophylactic purposes or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, inhibition of metastasis, slowing the rate of disease progression, amelioration or alleviation of pathology, and remission or improvement of prognosis. In some embodiments, the antigen-binding molecules according to the present disclosure are used to delay the onset of a disease or to delay the progression of a disease.

[0129] The term "effector function" refers to a biological activity attributable to the Fc region of an antibody or antigen-binding molecule according to the present disclosure, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and activation of B cells.

[0130] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies or antigen-binding molecules of the disclosure bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable such cytotoxic effector cells to specifically bind to antigen-bearing target cells and then kill the target cells with cytotoxins. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII.

[0131] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) or antigen-binding molecules of the disclosure.

[0132] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to the mechanism by which antibody- or antigen-binding molecule-coated target cells are eliminated by internalization by phagocytes such as macrophages or dendritic cells.

[0133] The terms "inhibit" or "inhibiting" or "reducing" or "reducing" or "neutralizing" or "neutralizing" refer to the reduction or cessation of any phenotypic characteristic (such as binding, biological activity or function), or the reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Inhibition", "reduction" or "neutralization" need not be complete, so long as it is detectable using an appropriate assay. In some embodiments, "reducing" or "inhibiting" refers to the ability to effect a reduction of 20% or more. In other embodiments, "reducing" or "inhibiting" refers to the ability to effect a reduction of 50% or more. In yet other embodiments, "reducing" or "inhibiting" refers to the ability to effect an overall reduction of 75%, 85%, 90%, 95%, or more.

[0134] As used herein, a "human antibody" or "human antibody fragment" includes antibodies and antibody fragments having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Additionally, if the antibody contains a constant region, the constant region is also derived from such sequences. Human origin includes, for example, antibodies that contain human germline sequences, or mutated versions of human germline sequences, or consensus framework sequences obtained from human framework sequence analysis, e.g., as described in Knappik et al., (2000) J Mol Biol 296:57-86). The structures and positions of immunoglobulin variable domains, e.g., CDRs, can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services (1991), eds. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948; Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 USDepartment of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 106:111-115). 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948. Human antibodies and human variable regions can also be isolated from synthetic libraries or transgenic mice (e.g., Xenomouse), provided that each system produces antibodies with variable regions in which both the framework and CDR regions are derived from sequences of human origin.

[0135] The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody having constant antibody regions derived from or corresponding to sequences present in one species and variable antibody regions derived from another species. Preferably, the constant antibody regions are derived from or correspond to sequences present in humans and the variable antibody regions (e.g. VH, VL, CDR or FR regions) are derived from sequences present in a non-human animal, such as mouse, rat, rabbit or hamster.

[0136] A "humanized antibody" or "humanized antibody fragment" is defined herein as an antibody molecule having constant antibody regions and variable antibody regions or portions thereof derived from sequences of human origin, or in which only the CDRs are derived from another species. Humanization can be achieved in a variety of ways, including but not limited to: (a) grafting non-human (e.g., donor antibody) CDRs into human (e.g., recipient antibody) framework and constant regions, with or without preserving key framework residues (e.g., residues important for preserving good antigen binding affinity or antibody function), (b) grafting only non-human specificity determining regions (SDRs or a-CDRs; residues important for antibody-antigen interaction) into human framework and constant regions, or (c) grafting entire non-human variable domains but "covering" them with human-like sections by replacement of surface residues.Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. 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, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing "resurfacing"); Dall'Acqua et al, Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn et al, Methods 36, 61-68 (2005) and Klimka et al, Br J Cancer 83, 252-260 (2000) (describing "guided selection" for FR shuffling).

[0137] The term "isolated" refers to a compound, such as an antibody, antibody fragment, or antigen-binding molecule, that is substantially free of other antibodies, antibody fragments, or antigen-binding molecules with different antigen specificities. Furthermore, an isolated antibody, antibody fragment, or antigen-binding molecule may be substantially free of other cellular material and / or chemicals. Thus, in some embodiments, an antibody, antibody fragment, or antigen-binding molecule provided herein is an isolated antibody, antibody fragment, or antigen-binding molecule that is separated from antibodies or antigen-binding molecules with different specificities. An isolated antibody or antigen-binding molecule may be a monoclonal antibody, antibody fragment, or antigen-binding molecule. An isolated antibody, antibody fragment, or antigen-binding molecule may be a recombinant monoclonal antibody, antibody fragment, or antigen-binding molecule. However, an isolated antibody, antibody fragment, or antigen-binding molecule that specifically binds to a target epitope, isoform, or variant may have cross-reactivity to other related antigens, such as antigens from other species (e.g., species homologs).

[0138] The term "recombinant antibody", "recombinant antibody fragment" or "recombinant antigen-binding molecule" as used herein includes any antibody, antibody fragment or antigen-binding molecule according to the present disclosure that is prepared, expressed, generated or isolated by non-naturally occurring means. For example, antibodies or antigen-binding molecules isolated from host cells transformed to express the antibody or antigen-binding molecule, antibodies selected and isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, generated or isolated by any other means including splicing all or part of a human immunoglobulin gene, sequence to other DNA sequences, or antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom. Preferably, such recombinant antibodies or antigen-binding molecules have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when transgenic animals of human Ig sequences are used) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, do not naturally occur within the human antibody germline repertoire in vivo. The recombinant antibody or antigen-binding molecule may be a recombinant monoclonal antibody or a recombinant monoclonal antigen-binding molecule. In one embodiment, the antibodies and antibody fragments disclosed herein are isolated from the Ylanthia® antibody library disclosed in US 13 / 321,564 or 13 / 299,367.

[0139] As used herein, the term "monoclonal antibody," "monoclonal antibody fragment," or "monoclonal antigen-binding molecule" refers to an antibody, antibody fragment, or antigen-binding molecule disclosed herein that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies or antibody fragments can be produced by the hybridoma method as described in Kohler et al.; Nature, 256:495 (1975), or isolated from a phage library. Other methods for preparing clonal cell lines and the monoclonal antibodies or antigen-binding molecules expressed thereby disclosed herein are known in the art (see, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York).

[0140] A "therapeutically effective amount" or "effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the side effects of a disease.

[0141] "Administered" or "administration" includes, but is not limited to, delivery of an agent in an injectable form, such as, for example, intravenous, intramuscular, intradermal or subcutaneous routes, or mucosal routes, such as a nasal spray or aerosol for inhalation, or as an ingestible solution, capsule or tablet. Preferably, administration is in an injectable form.

[0142] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0143] "Subject" or "species" or as used in this context refers to any mammal, including rodents, such as mice or rats, and primates, such as cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), or humans (Homo sapiens). Preferably, the subject is a primate, and most preferably a human.

[0144] A "wild-type" protein is a version or variant of a protein that occurs in nature. The amino acid sequence of a wild-type protein, such as the Fc region of a human IgG1 antibody, is the amino acid sequence of a protein that occurs in nature. Due to allotype differences, there may be more than one amino acid sequence of a wild-type protein. For example, there are several allotypes of the naturally occurring human IGg1 heavy chain constant region (see, for example, Jeffries et al. (2009) mAbs 1:1).

[0145] As used herein, "IC 50 " refers to the concentration of a set of antigen-binding molecules that inhibits a response halfway between the maximum response and the baseline in an assay. This represents the concentration of the set of antigen-binding molecules that suppresses a given response by 50%.

[0146] As used herein, "non-covalent bonds" refer to interactions between molecules that do not involve bonds between atoms. Non-covalent interactions include, for example, ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces.

[0147] As used herein, "covalent bond" refers to a bond between atoms characterized by the sharing of electrons.

[0148] As used herein, the term "about," when used in connection with a specific stated numerical value, means that the value may vary from the stated value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0149] Embodiment Provided herein is a combination or set of a first antigen-binding molecule and a second antigen-binding molecule, where each of the two antigen-binding molecules is composed of a targeting moiety having specificity for a target antigen or antigen epitope fused via a peptide linker to either the unpaired VL or VH domain of an antibody Fv domain specific for a T cell antigen, e.g., CD3. The unpaired (or split) VL or VH domain present in each of the two antigen-binding molecules cannot bind to a T cell antigen alone. However, when both antigen-binding molecules bind to their antigen or antigen epitope expressed on the surface of a cell via their targeting moieties, the unpaired VL and VH domains come into close proximity and interact with each other to reconstitute the original antibody Fv domain. The trispecific heterodimeric antibody molecule thus formed on-cell can induce and stimulate T cells for the destruction of the target cell if the reassembled or newly formed Fv domain is specific for CD3.

[0150] In one embodiment, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a) from its N-terminus to its C-terminus: i. a first targeting moiety specific for a first antigen or a first antigen epitope; ii. a first peptide linker, and iii. either the VH or VL domain of an antibody Fv domain specific for a second antigen or a second antigen epitope; A first antigen-binding molecule consisting of or comprising: b) from its N-terminus to its C-terminus: i. a second targeting moiety specific for a third antigen or antigenic epitope; ii. a second peptide linker, and iii. A complementary VH or VL domain of an antibody Fv domain specific for a second antigen or antigenic epitope. A second antigen-binding molecule consisting of or comprising: The present invention provides a set of antigen-binding molecules consisting of or comprising:

[0151] In one embodiment, the first and second antigen-binding molecules are not covalently linked. In one embodiment, the first and second antigen-binding molecules are not covalently linked.

[0152] In one embodiment of the present disclosure, either the VH or VL domain of the antibody Fv domain of the first antigen-binding molecule and the complementary VH or VL domain of the second antigen-binding molecule can be non-covalently bound, thereby forming an antibody Fv domain specific for a second antigen or a second antigen epitope. In one embodiment, the non-covalent binding of the VH and VL domains results in functional complementarity of the antibody Fv domain specific for a second antigen or a second antigen epitope. In one embodiment, the non-covalent binding of the VH and VL domains results in the formation of an antibody Fv domain specific for a second antigen or a second antigen epitope. In one embodiment, the antibody Fv domain is specific for CD3. In one embodiment, the second antigen is CD3, preferably CD3ε.

[0153] In one embodiment of the present disclosure, the non-covalent binding of the VH and VL domains and / or the formation of an antibody Fv domain specific to a second antigen or a second antigen epitope preferably occurs when the first and second antigen-binding molecules bind to their target antigens on the same cell and the complementary VL and VH domains come into close proximity. In one embodiment of the present disclosure, the non-covalent binding of the VH and VL domains selectively occurs when a cell expressing the first antigen or the first antigen epitope and the third antigen or the third antigen epitope is present. In one embodiment, the non-covalent binding of the VH and VL domains selectively occurs when a cell expressing the first antigen or the first antigen epitope and the third antigen or the third antigen epitope is present and both antigen-binding molecules are bound to the antigen or antigen epitope on the cell. In one embodiment, the non-covalent binding causes the first antigen-binding molecule and the third antigen-binding molecule to dimerize, forming a trispecific heteromeric antibody.

[0154] In one embodiment, the formation of the trispecific heteromeric antibody molecule occurs on a cell. In one embodiment, the formation of the trispecific heteromeric antibody molecule occurs in vivo. In one embodiment, the formation of the trispecific heteromeric antibody molecule occurs in vitro. In one embodiment, the formation of the trispecific heteromeric antibody molecule occurs in the presence of the first antigen or first antigen epitope and the third antigen or third antigen epitope.

[0155] In one embodiment, the on-cell formed trispecific heterodimeric antibody has monovalent binding to a first antigen or a first antigen epitope, monovalent binding to a third antigen or a third antigen epitope, and monovalent binding to a second antigen or a second antigen epitope.

[0156] In one embodiment, the on-cell forming trispecific heterodimeric antibody has monovalent binding to a first antigen or a first antigen epitope, bivalent binding to a third antigen or a third epitope, and monovalent binding to a second antigen or a second antigen epitope.

[0157] In one embodiment of the present disclosure, the on-cell formed trispecific heterodimeric antibody can induce and stimulate cytotoxic T cells for the destruction of target cells. In one embodiment, the on-cell formed trispecific antibody allows the formation of an immune synapse between a target cell and a cytotoxic T cell expressing CD3. In one embodiment, the on-cell formed trispecific antibody crosslinks the T cell with a target cell expressing the first and third antigens or antigenic epitopes, respectively, thereby forming an immune synapse and allowing the cytotoxic T cell to kill the target cell. In one embodiment, the width of the immune synapse formed between the target cell and the T cell is about 200 Å, about 190 Å, about 180 Å, about 170 Å, about 160 Å, about 150 Å, about 140 Å, about 130 Å, about 120 Å, about 110 Å, about 100 Å, about 90 Å, about 80 Å, about 70 Å, about 60 Å, about 50 Å, about 40 Å, about 30 Å, about 20 Å, or about 10 Å.

[0158] In one embodiment, the immune synapse is formed by the presence of the first and second antigen-binding molecules included in the set of antigen-binding molecules according to the present disclosure. In one embodiment of the present disclosure, the efficacy of the on-cell forming trispecific antibody in mediating the killing of target cells depends on the width of the formed immune synapse. In one embodiment, the efficacy of the on-cell trispecific antibody in mediating the killing of target cells increases inversely proportional to the width of the formed immune synapse.

[0159] Thus, in one embodiment, the efficacy of the on-cell formed trispecific antibody in mediating target cell killing depends on the distance of the binding site of the first targeting moiety to the binding site of the newly formed antibody Fv domain and the distance of the binding site of the second targeting moiety to the binding site of the newly formed antibody Fv domain.

[0160] In a further embodiment, the efficacy of the on-cell forming trispecific antibody in mediating killing of a target cell depends on the first antigen or first antigen epitope and the second antigen or second antigen epitope.

[0161] In a specific embodiment, in the set of antigen-binding molecules according to the present disclosure, the first and second antigen-binding molecules are selected based on the first antigen or the first antigen epitope and the second antigen or the second antigen epitope, in particular based on the distance from the first antigen epitope to the cell surface and the distance from the second antigen epitope to the cell surface, and / or the distance from the first antigen or antigen epitope to the second antigen or antigen epitope.

[0162] (1 1 / 2) Antigen-binding molecules of B027 format and their combinations In one embodiment, the present disclosure provides a polypeptide comprising, from its N-terminus to its C-terminus: a) a first targeting moiety specific for a first antigen or a first antigen epitope; b) a peptide linker, and c) either the VH or VL domain of a second binding site specific for a second antigen. An antigen-binding molecule consisting of or comprising: The present invention relates to an antigen-binding molecule in which a targeting moiety is fused via a peptide linker to the N-terminus of either the VH or VL domain of a second binding site specific for a second antigen.

[0163] In one embodiment, the targeting moiety is fused via a peptide linker to the N-terminus of the VH domain of the second binding site specific for the second antigen. In one embodiment, the targeting moiety is fused via a peptide linker to the N-terminus of the VL domain of the second binding site specific for the second antigen.

[0164] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab.

[0165] In one embodiment, the second binding site is an Fv domain. In one embodiment, the second binding site consists of an Fv domain. In one embodiment, the second binding site is comprised in an Fv domain. In one embodiment, the second binding site comprises an Fv domain. In one embodiment, the antigen binding molecule comprises either the VH domain or the VL domain of the second binding site, but not both variable domains of the second binding site. In one embodiment, if the antigen binding molecule comprises a VH domain of the second binding site, it does not comprise a VL domain of the second binding site, or if the antigen binding molecule comprises a VL domain of the second binding site, it does not comprise a VH domain of the second binding site. In one embodiment, the VH domain and the VL domain of the second binding site form an Fv domain.

[0166] In one embodiment, the present disclosure provides a polypeptide comprising, from its N-terminus to its C-terminus: a) a Fab comprising a first binding site specific for a first antigen or a first antigen epitope; b) a peptide linker, and c) either the VH or VL domain of a second binding site specific for a second antigen. An antigen-binding molecule consisting of or comprising: The present invention relates to an antigen-binding molecule in which the C-terminus of a Fab heavy chain is fused to the N-terminus of either the VH or VL domain of a second binding site via a peptide linker.

[0167] In one embodiment, the C-terminus of the Fab heavy chain is fused via a peptide linker to the N-terminus of the VH domain of a second binding site specific for a second antigen, hi one embodiment, the C-terminus of the Fab heavy chain is fused via a peptide linker to the N-terminus of the VL domain of the second binding site.

[0168] In one embodiment of the present disclosure, an antigen binding molecule according to the present disclosure has a structure as depicted in FIG. 1A.

[0169] In one embodiment of the present disclosure, an antigen binding molecule according to the present disclosure consists of or comprises two polypeptides, wherein: a) the first polypeptide consists of or comprises a light chain of a Fab; b) the second polypeptide comprises from its N-terminus to its C-terminus: the heavy chain of i.Fab, ii. a peptide linker, and iii. either the VH domain or the VL domain of a second binding site specific for a second antigen. It consists of or includes:

[0170] In one embodiment of the present disclosure, the present disclosure provides: a) from its N-terminus to its C-terminus: i. a first targeting moiety comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. A first antigen-binding molecule consisting of or comprising: a first antigen-binding molecule in which a targeting moiety is fused to the N-terminus of either the VH or VL domain of the second binding site via a first peptide linker; b) from its N-terminus to its C-terminus: i. a second targeting moiety comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a second peptide linker; iii. a complementary VH or VL domain of a second binding site specific for a second antigen. A second antigen-binding molecule consisting of or comprising: A second antigen-binding molecule, in which a second targeting moiety is fused to the N-terminus of the complementary VH or VL domain of the second binding site via a second peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0171] In one embodiment, the first targeting moiety is fused to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker. In one embodiment, the first targeting moiety is fused to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker.

[0172] In one embodiment, the second targeting moiety is fused to the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker. In one embodiment, the second targeting moiety is fused to the N-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker.

[0173] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab.

[0174] In one embodiment of the present disclosure, the present disclosure provides: a) from its N-terminus to its C-terminus: i. a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. A first antigen-binding molecule consisting of or comprising: A first antigen-binding molecule in which the C-terminus of a first Fab heavy chain is fused to the N-terminus of either the VH or VL domain of a second binding site via a first peptide linker; b) from its N-terminus to its C-terminus: i. a second Fab comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a second peptide linker, and iii. a complementary VH or VL domain of a second binding site specific for a second antigen. A second antigen-binding molecule consisting of or comprising: A second antigen-binding molecule in which the C-terminus of the second Fab heavy chain is fused to the N-terminus of the complementary VH or VL domain of the second binding site via a second peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0175] In one embodiment, the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen. In one embodiment, the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen.

[0176] In one embodiment, the C-terminus of the second Fab heavy chain is fused via a second peptide linker to the N-terminus of the complementary VH domain of the second antigen binding molecule of the second binding site specific for the second antigen. In one embodiment, the C-terminus of the second Fab heavy chain is fused via a second peptide linker to the N-terminus of the complementary VL domain of the second antigen binding molecule of the second binding site specific for the second antigen.

[0177] In one embodiment of the present disclosure, the first antigen-binding molecule consists of or comprises a first and a second polypeptide, wherein: a) the first polypeptide consists of or comprises the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. It consists of or includes:

[0178] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a third and a fourth polypeptide, wherein: a) the third polypeptide consists of or comprises the light chain of a second Fab; b) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a second peptide linker, and iii. either a complementary VH or VL domain of a second binding site specific for a second antigen; It consists of or includes:

[0179] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH and VL domains of the second binding molecule can be non-covalently linked to form a second binding site.

[0180] In one embodiment, the present disclosure relates to a set of antigen-binding molecules consisting of or including a first antigen-binding molecule having a structure as depicted in FIG. 1A and a second antigen-binding molecule having a structure as depicted in FIG. 1A.

[0181] (1 1 / 2)B036 Antigen-binding molecules in Fc-KiH format and combinations thereof In one embodiment, the present disclosure provides a polypeptide comprising, from its N-terminus to its C-terminus: a) a targeting moiety comprising a first binding site specific for a first antigen or a first antigenic epitope; b) a first peptide linker; c) either the VH or VL domain of a second binding site specific for a second antigen; d) a second peptide linker, and e) a first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; In an antigen-binding molecule comprising or consisting of: the targeting moiety is fused to the N-terminus of either the VH or VL domain of the second binding site region via a first peptide linker; the C-terminus of the VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker; and The N-terminus of the second Fc region subunit is fused to a third peptide linker, related to the antigen-binding molecule.

[0182] In one embodiment, the targeting moiety is fused via a first peptide linker to the N-terminus of the VH domain of the second binding site specific for the second antigen. In one embodiment, the targeting moiety is fused via a first peptide linker to the N-terminus of the VL domain of the second binding site specific for the second antigen.

[0183] In one embodiment, the C-terminus of the VH domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker. In one embodiment, the C-terminus of the VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker.

[0184] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab.

[0185] In one embodiment, the present disclosure provides a polypeptide comprising, from its N-terminus to its C-terminus: a) a Fab comprising a first binding site specific for a first antigen or a first antigen epitope; b) a first peptide linker; c) either the VH or VL domain of a second binding site specific for a second antigen; d) a second peptide linker, and e) a first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; In an antigen-binding molecule comprising or consisting of: the C-terminus of the Fab heavy chain is fused via a first peptide linker to the N-terminus of either the VH or VL domain of a second binding site region; the C-terminus of the VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker; and The N-terminus of the second Fc region subunit is fused to a third peptide linker, related to the antigen-binding molecule.

[0186] In one embodiment, the C-terminus of the Fab heavy chain is fused via a first peptide linker to the N-terminus of the VH domain of a second binding site specific for a second antigen. In one embodiment, the C-terminus of the Fab heavy chain is fused via a first peptide linker to the N-terminus of the VL domain of a second binding site specific for a second antigen.

[0187] In one embodiment, the C-terminus of the VH domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker. In one embodiment, the C-terminus of the VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker.

[0188] In one embodiment of the disclosure, the antigen binding molecule consists of or comprises three polypeptides, wherein: a) the first polypeptide consists of or comprises a light chain of a Fab; b) the second polypeptide comprises from its N-terminus to its C-terminus: the heavy chain of i.Fab, ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker, and v. a first Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; and c) the third polypeptide comprises from its N-terminus to its C-terminus: i. a third peptide linker, and ii. a second Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus It consists of:

[0189] In one embodiment, the antigen binding molecule has a structure as depicted in FIG. 1B.

[0190] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first targeting moiety comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker; v. A first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. In the first antigen-binding molecule consisting of or comprising: the targeting moiety is fused to the N-terminus of either the VH or VL domain of the second binding site via a first peptide linker; the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker; and A first antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a third peptide linker; b) from its N-terminus to its C-terminus: i. a second targeting moiety comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a fourth peptide linker; iii. a complementary VH or VL domain of a second binding site specific for a second antigen; iv. a fifth peptide linker; v. A second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. In the second antigen-binding molecule consisting of or comprising: a second targeting moiety is fused to the N-terminus of either the VH or VL domain of the second binding site via a fourth peptide linker; the C-terminus of the complementary VH or VL domain of the second binding site is fused to the N-terminus of a third Fc region subunit via a fifth peptide linker; and A second antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a sixth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0191] In one embodiment, the first targeting moiety is fused to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker. In one embodiment, the first targeting moiety is fused to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker.

[0192] In one embodiment, the second targeting moiety is fused to the N-terminus of the complementary VH domain of the second antigen binding molecule of the second binding site specific for the second antigen via a fourth peptide linker. In one embodiment, the second targeting moiety is fused to the N-terminus of the complementary VL domain of the second antigen binding molecule of the second binding site specific for the second antigen via a fourth peptide linker.

[0193] In one embodiment, the C-terminus of the VH domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker. In one embodiment, the C-terminus of the VL domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker.

[0194] In one embodiment, the C-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site is fused to the N-terminus of the third Fc region subunit via a fifth peptide linker. In one embodiment, the C-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site is fused to the N-terminus of the third Fc region subunit via a sixth peptide linker.

[0195] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of a Fab, a scFab, a Fab', a scFv, a dsFv, and a single domain antibody. In one embodiment, the targeting moiety is a Fab.

[0196] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker, and v. A first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. In the first antigen-binding molecule consisting of or comprising: the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of either the VH or VL domain of the second binding site; the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker; and A first antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a third peptide linker; b) from its N-terminus to its C-terminus: i. a second Fab comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a fourth peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a fifth peptide linker, and v. A second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. In the second antigen-binding molecule consisting of or comprising: the C-terminus of the second Fab heavy chain is fused via a fourth peptide linker to the N-terminus of either the complementary VH or VL domain of the second binding site; the C-terminus of the VH or VL domain of the second binding site is fused to the N-terminus of a third Fc region subunit via a fifth peptide linker; and The N-terminus of the second Fc region subunit is fused to a sixth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0197] In one embodiment, the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen. In one embodiment, the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen.

[0198] In one embodiment, the C-terminus of the second Fab heavy chain is fused via a fourth peptide linker to the N-terminus of the complementary VH domain of the second antigen binding molecule of the second binding site specific for the second antigen. In one embodiment, the C-terminus of the second Fab heavy chain is fused via a fourth peptide linker to the N-terminus of the complementary VL domain of the second antigen binding molecule of the second binding site specific for the second antigen.

[0199] In one embodiment, the C-terminus of the VH domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker. In one embodiment, the C-terminus of the VL domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker.

[0200] In one embodiment, the C-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site is fused to the N-terminus of the third Fc region subunit via a fifth peptide linker. In one embodiment, the C-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site is fused to the N-terminus of the third Fc region subunit via a fifth peptide linker.

[0201] In one embodiment of the disclosure, the antigen binding molecule consists of or comprises a first, second and third polypeptide, wherein: a) the first polypeptide consists of or comprises the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker, and v. a first Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; and c) the third polypeptide comprises from its N-terminus to its C-terminus: i. a third peptide linker, and ii. a second Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus It consists of:

[0202] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a fourth, fifth and sixth polypeptide, wherein: a) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. a sixth peptide linker, and ii. from its N-terminus to its C-terminus, a fourth Fc region subunit composed of a CH2 and a CH3 domain. consisting of or comprising: b) the fifth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a fourth peptide linker; iii. either a complementary VH or VL domain of a second binding site specific for a second antigen; iv. a fifth peptide linker, and v. a third Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; consisting of or comprising: c) the sixth polypeptide comprises the light chain of the second Fab.

[0203] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH domain and the VL domain of the second binding site can be non-covalently linked to form the second binding site.

[0204] In one embodiment, the present disclosure relates to a set of antigen-binding molecules consisting of or including a first antigen-binding molecule having a structure as depicted in FIG. 1B and a second antigen-binding molecule having a structure as depicted in FIG. 1B.

[0205] (1 1 / 2)B038 Fc-KiH format antigen-binding molecules and combinations thereof In one embodiment, the present disclosure provides a polypeptide comprising, from its N-terminus to its C-terminus: a) a targeting moiety comprising a first binding site specific for a first antigen or a first antigenic epitope; b) a first peptide linker; c) an Fc region composed of a first and a second Fc region subunit, each of which is composed of a CH2 and a CH3 domain; d) a second peptide linker, and e) either the VH or VL domain of a second binding site specific for a second antigen. An antigen-binding molecule consisting of or comprising: the targeting moiety is fused to the N-terminus of either the VH or VL domain of the first Fc region subunit via a first peptide linker; the N-terminus of either the VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a second peptide linker; and The N-terminus of the second Fc region subunit is fused to a third peptide linker, related to the antigen-binding molecule.

[0206] In one embodiment, the first Fc region subunit is fused via a second peptide linker to the N-terminus of the VH domain of a second binding site specific for a second antigen. In one embodiment, the first Fc region subunit is fused via a second peptide linker to the N-terminus of the VL domain of a second binding site specific for a second antigen.

[0207] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab.

[0208] In one embodiment, the present disclosure provides a polypeptide comprising, from its N-terminus to its C-terminus: a) a Fab comprising a first binding site specific for a first antigen or a first antigen epitope; b) a first peptide linker; c) an Fc region composed of a first and a second Fc region subunit, each of which is composed of a CH2 and a CH3 domain; d) a second peptide linker, and e) either the VH or VL domain of a second binding site specific for a second antigen. In an antigen-binding molecule comprising or consisting of: the C-terminus of the Fab heavy chain is fused to the N-terminus of a first Fc region subunit via a first peptide linker; the N-terminus of either the VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a second peptide linker; and The N-terminus of the second Fc region subunit is fused to a third peptide linker, related to the antigen-binding molecule.

[0209] In one embodiment, the first Fc region subunit is fused to the N-terminus of the VH domain of the second binding site via a second peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the VL domain of the second binding site via a second peptide linker.

[0210] In one embodiment of the disclosure, the antigen binding molecule consists of or comprises three polypeptides, wherein: a) the first polypeptide consists of the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. a first Fc region subunit composed of, from its N-terminus to its C-terminus, a CH2 and a CH3 domain; iv. a second peptide linker, and v. Either the VH or VL domain of the second binding site It consists of c) the third polypeptide comprises from its N-terminus to its C-terminus: i. a third peptide linker, and ii. From its N-terminus to its C-terminus, it consists of a second Fc region subunit composed of a CH2 and a CH3 domain.

[0211] In one embodiment, an antigen binding molecule according to the present disclosure has a structure as depicted in FIG. 1C.

[0212] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a)Below: i. a first targeting moiety comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker; iii. a first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; iv. a second peptide linker, and v. either the VH or VL domain of a second binding site specific for a second antigen In the first antigen-binding molecule consisting of or comprising: the targeting moiety is fused to the N-terminus of the first Fc region subunit via a first peptide linker; the N-terminus of the VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a second peptide linker; A first antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a third peptide linker; b)Below: i. a second targeting moiety comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a fourth peptide linker; iii. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; iv. a fifth peptide linker; v. A complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: the targeting moiety is fused to the N-terminus of the third Fc region subunit via a fourth peptide linker; the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of a third Fc region subunit via a fifth peptide linker; and the N-terminus of the fourth Fc region subunit is fused to a sixth peptide linker; The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0213] In one embodiment, the first Fc region subunit is fused to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker.

[0214] In one embodiment, the C-terminus of the third Fc region subunit is fused to the N-terminus of the complementary VH domain of the second antigen binding molecule of the second binding site specific for the second antigen via a fifth peptide linker. In one embodiment, the C-terminus of the third Fc region subunit is fused to the N-terminus of the VL domain of the second antigen binding molecule of the second binding site specific for the second antigen via a fifth peptide linker.

[0215] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH domain and the VL domain of the second binding can be non-covalently linked to form a second binding site.

[0216] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab.

[0217] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a)Below: i. a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker; iii. a first Fc region composed of a first and a second Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; iv. a second peptide linker, and v. either the VH or VL domain of a second binding site specific for a second antigen In the first antigen-binding molecule, which consists of or comprises: the C-terminus of the first Fab heavy chain is fused to the N-terminus of the first Fc region subunit via a first peptide linker; the N-terminus of either the VH or VL of the second binding site is fused to the C-terminus of the first Fc region subunit via a second peptide linker; A first antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a third peptide linker; b)Below: i. a second Fab comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a fourth peptide linker; iii. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; iv. a fifth peptide linker; v. A complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: the C-terminus of the second Fab heavy chain is fused to the N-terminus of a third Fc region subunit via a fourth peptide linker; the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of a third Fc region subunit via a fifth peptide linker; and a second antigen-binding molecule, wherein the N-terminus of the fourth Fc region subunit is fused to a sixth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0218] In one embodiment, the C-terminus of the first Fc region subunit is fused to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker. In one embodiment, the C-terminus of the first Fc region subunit is fused to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker.

[0219] In one embodiment, the C-terminus of the third Fc region subunit is fused to the N-terminus of the complementary VH domain of the second antigen binding molecule of the second binding site specific for the second antigen via a fifth peptide linker. In one embodiment, the C-terminus of the third Fc region subunit is fused to the N-terminus of the VL domain of the second antigen binding molecule of the second binding site specific for the second antigen via a fifth peptide linker.

[0220] In one embodiment of the present disclosure, the first antigen-binding molecule consists of or comprises a first, second and third polypeptide, wherein: a. the first polypeptide consists of a light chain of a first Fab; b. the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. a first Fc region subunit composed of, from its N-terminus to its C-terminus, a CH2 and a CH3 domain; iv. a second peptide linker, and v. Either the VH or VL domain of the second binding site It consists of c. the third polypeptide comprises from its N-terminus to its C-terminus: i. a third peptide linker, and ii. a second Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus It consists of:

[0221] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a fourth, fifth and sixth polypeptide, wherein: a. the fourth polypeptide comprises from its N-terminus to its C-terminus: i. a sixth peptide linker, and ii. from its N-terminus to its C-terminus, a fourth Fc region subunit composed of a CH2 and a CH3 domain. It consists of b. the fifth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a fourth peptide linker; iii. a third Fc region subunit composed of a CH2 and a CH3 domain from its N-terminus to its C-terminus; iv. a fifth peptide linker, and v. either the complementary VH or VL domain of the second binding site It consists of c. The sixth polypeptide consists of or comprises the light chain of the second Fab.

[0222] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH and VL domains of the second binding site can be non-covalently linked to form the second binding site.

[0223] In one embodiment, the present disclosure relates to a set of antigen-binding molecules consisting of or including a first antigen-binding molecule having a structure as depicted in FIG. 1C and a second antigen-binding molecule having a structure as depicted in FIG. 1C.

[0224] (2 1 / 2)B064 Fc-KiH format antigen-binding molecules and combinations thereof In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) a first targeting moiety comprising a first binding site specific for a first antigen or a first antigenic epitope; b) a second targeting moiety comprising a third binding site specific for the first antigen or an epitope of the first antigen; c) a first peptide linker; d) a third peptide linker; e) an Fc region composed of a first and a second Fc region subunit, each of which is composed of a CH2 and a CH3 domain; f) a second peptide linker, and g) either the VH or VL domain of a second binding site specific for a second antigen. An antigen-binding molecule consisting of or comprising: a first targeting moiety is fused to the N-terminus of the first Fc region subunit via a first peptide linker; a second targeting moiety is fused to the N-terminus of the second Fc region subunit via a third peptide linker; The present invention relates to an antigen-binding molecule in which the N-terminus of either the VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a second peptide linker.

[0225] In one embodiment, the first Fc region subunit is fused via a second peptide linker to the N-terminus of the VH domain of a second binding site specific for a second antigen. In one embodiment, the first Fc region subunit is fused via a second peptide linker to the N-terminus of the VL domain of a second binding site specific for a second antigen.

[0226] In one embodiment, a first targeting moiety is fused at its C-terminus to the N-terminus of a first Fc region subunit via a first peptide linker, and a second targeting moiety is fused at its C-terminus to the N-terminus of a second Fc region subunit via a third peptide linker.

[0227] In one embodiment, the first targeting moiety and the second targeting moiety are the same.

[0228] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab. In one embodiment, the first peptide linker and the second peptide linker are the same.

[0229] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; b) a second Fab comprising a third binding site specific for the first antigen or an epitope of the first antigen; c) a first peptide linker; d) a third peptide linker; c) an Fc region composed of a first and a second Fc region subunit, each of which is composed of a CH2 and a CH3 domain; d) a second peptide linker, and e) either the VH or VL domain of a second binding site specific for a second antigen. An antigen-binding molecule consisting of or comprising: the C-terminus of the first Fab heavy chain is fused to the N-terminus of the first Fc region subunit via a first peptide linker; the C-terminus of the second Fab heavy chain is fused to the N-terminus of a second Fc region subunit via a third peptide linker; The N-terminus of either the VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a second peptide linker.

[0230] In one embodiment, the first Fc region subunit is fused to the N-terminus of the VH domain of the second binding site via a second peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the VL domain of the second binding site via a second peptide linker.

[0231] In one embodiment of the disclosure, the antigen binding molecule consists of or comprises three polypeptides, wherein: a) the first polypeptide consists of or comprises the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. a first Fc region subunit composed of, from its N-terminus to its C-terminus, a CH2 and a CH3 domain; iv. a second peptide linker, and v. Either the VH or VL domain of the second binding site It consists of c) the third polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a third peptide linker, and iii. from its N-terminus to its C-terminus, a second Fc region subunit composed of a CH2 and a CH3 domain; d) the fourth polypeptide consists of or comprises the light chain of the second Fab.

[0232] In one embodiment, an antigen binding molecule according to the present disclosure has a structure as depicted in FIG. 4A.

[0233] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a)Below: i. a first targeting moiety comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a second targeting moiety comprising a third binding site specific for the first antigen or an epitope of the first antigen; iii. a first peptide linker; iv. a third peptide linker; v. an Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; vi. a second peptide linker, and vii. either the VL or VH domain of a second binding site specific for a second antigen; In the first antigen-binding molecule consisting of or comprising: a first targeting moiety fused to the N-terminus of the first Fc region subunit via a first peptide linker; a second targeting moiety is fused to the N-terminus of the second Fc region subunit via a third peptide linker; A first antigen-binding molecule, in which the N-terminus of either the VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a second peptide linker; b)Below: i. a third targeting moiety comprising a fourth binding site specific for a third antigen or a third antigen epitope; ii. a fourth targeting moiety comprising a fifth binding site specific for a third antigen or a third antigen epitope; iii. a fourth peptide linker; iv. a sixth peptide linker; v. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; vi. a fifth peptide linker; vii. a complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: a third targeting moiety is fused to the N-terminus of the third Fc region subunit via a fourth peptide linker; a fourth targeting moiety is fused to the N-terminus of the fourth Fc region subunit via a sixth peptide linker; A second antigen-binding molecule, wherein the N-terminus of either the VH or VL domain of the second binding site is fused to the C-terminus of a third Fc region subunit via a fifth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0234] In one embodiment, the C-terminus of the first Fc region subunit is fused to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker.

[0235] In one embodiment, the C-terminus of the third Fc region subunit is fused to the N-terminus of the complementary VH domain of the second antigen binding molecule of the second binding site specific for the second antigen via a fifth peptide linker. In one embodiment, the C-terminus of the third Fc region subunit is fused to the N-terminus of the VL domain of the second antigen binding molecule of the second binding site specific for the second antigen via a fifth peptide linker.

[0236] In one embodiment, a first targeting moiety is fused at its C-terminus to the N-terminus of a first Fc region subunit via a first peptide linker, a second targeting moiety is fused at its C-terminus to the N-terminus of a second Fc region subunit via a third peptide linker, a third targeting moiety is fused at its C-terminus to the N-terminus of a third Fc region subunit via a fourth peptide linker, and a fourth targeting moiety is fused at its C-terminus to the N-terminus of a fourth Fc region subunit via a sixth peptide linker.

[0237] In one embodiment, the first targeting moiety and the second targeting moiety are the same. In one embodiment, the third targeting moiety and the fourth targeting moiety are the same. In one embodiment, the targeting moiety is an antibody or an antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab.

[0238] In an embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In an embodiment of the present disclosure, the VH domain and the VL domain of the second binding can be non-covalently linked to form a second binding site.

[0239] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a)Below: i. a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a second Fab comprising a third binding site specific for the first antigen or an epitope of the first antigen; iii. a first peptide linker; iv. a third peptide linker; v. an Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; vi. a second peptide linker, and vii. either the VL or VH domain of a second binding site specific for a second antigen; In the first antigen-binding molecule consisting of or comprising: the C-terminus of the first Fab heavy chain is fused to the N-terminus of the first Fc region subunit via a first peptide linker; the C-terminus of the second Fab heavy chain is fused to the N-terminus of a second Fc region subunit via a third peptide linker; A first antigen-binding molecule, in which the N-terminus of either the VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a second peptide linker; b)Below: i. a third Fab comprising a fourth binding site specific for a third antigen or a third antigen epitope; ii. a fourth Fab comprising a fifth binding site specific for a third antigen or a third antigen epitope; iii. a fourth peptide linker; iv. a sixth peptide linker; v. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; vi. a fifth peptide linker; vii. a complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: the C-terminus of the third Fab heavy chain is fused via a fourth peptide linker to the N-terminus of a third Fc region subunit; the C-terminus of the fourth Fab heavy chain is fused to the N-terminus of the fourth Fc region subunit via a sixth peptide linker; A second antigen-binding molecule, wherein the N-terminus of either the VH or VL domain of the second binding site is fused to the C-terminus of a third Fc region subunit via a fifth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0240] In one embodiment, the first Fc region subunit is fused at its C-terminus to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker. In one embodiment, the first Fc region subunit is fused at its C-terminus to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker.

[0241] In one embodiment, the third Fc region subunit is fused at its C-terminus to the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a fifth peptide linker. In one embodiment, the third Fc region subunit is fused at its C-terminus to the N-terminus of the VL domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a fifth peptide linker.

[0242] In one embodiment of the present disclosure, the first antigen binding molecule consists of or comprises a first, second, third and fourth polypeptide, wherein: a) the first polypeptide consists of or comprises the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. a first Fc region subunit composed of, from its N-terminus to its C-terminus, a CH2 and a CH3 domain; iv. a second peptide linker, and v. Either the VH or VL domain of the second binding site It consists of c) the third polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a third peptide linker, and iii. from its N-terminus to its C-terminus, a second Fc region subunit composed of a CH2 and a CH3 domain; d) the fourth polypeptide consists of or comprises the light chain of the second Fab.

[0243] In one embodiment of the present disclosure, the second antigen binding molecule consists of or comprises a fourth, fifth, sixth and seventh polypeptide, a) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a fourth Fab, ii. a sixth peptide linker, and iii. from its N-terminus to its C-terminus, a fourth Fc region subunit composed of a CH2 and a CH3 domain. Including, b) the fifth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a third Fab; ii. a fourth peptide linker; iii. a third Fc region subunit composed of a CH2 and a CH3 domain from its N-terminus to its C-terminus; iv. a fifth peptide linker, and v. The complementary VH or VL domain of the second binding site Including, c) the sixth polypeptide consists of or comprises the light chain of a third Fab; d) the seventh polypeptide consists of or comprises the light chain of the fourth Fab.

[0244] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH and VL domains of the second binding molecule can be non-covalently linked to form a second binding site.

[0245] In one embodiment, the present disclosure relates to a set of antigen-binding molecules consisting of or including a first antigen-binding molecule having a structure as depicted in FIG. 4A and a second antigen-binding molecule having a structure as depicted in FIG. 4A.

[0246] Set of antigen-binding molecules in (1 1 / 2)B027 format and (1 1 / 2)B036 Fc-KiH format In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first targeting moiety comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. A first antigen-binding molecule consisting of or comprising: A first antigen-binding molecule in which a first targeting moiety is fused to the N-terminus of either the VH or VL domain of a second binding site specific for a second antigen via a first peptide linker; b) from its N-terminus to its C-terminus: i. a second targeting moiety comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a second peptide linker; iii. a complementary VH or VL domain of a second binding site specific for a second antigen; iv. a third peptide linker; v. A first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. In the second antigen-binding molecule, which consists of or comprises: a second targeting moiety is fused to the N-terminus of the complementary VH or VL domain of the second binding site via a second peptide linker; the C-terminus of the complementary VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a third peptide linker; and A second antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a fourth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0247] In one embodiment, the first targeting moiety is fused to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker. In one embodiment, the first targeting moiety is fused to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker.

[0248] In one embodiment, the second targeting moiety is fused to the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker. In one embodiment, the second targeting moiety is fused to the N-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a second peptide linker.

[0249] In one embodiment, the C-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a third peptide linker. In one embodiment, the C-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a third peptide linker.

[0250] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. A first antigen-binding molecule consisting of or comprising: A first antigen-binding molecule in which the C-terminus of a first Fab heavy chain is fused to the N-terminus of either the VH or VL domain of a second binding site specific for a second antigen via a first peptide linker; b) from its N-terminus to its C-terminus: i. a second Fab comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a second peptide linker; iii. a complementary VH or VL domain of a second binding site specific for a second antigen; i. a third peptide linker; ii. a first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; In the second antigen-binding molecule, which consists of or comprises: the C-terminus of the second Fab heavy chain is fused via a second peptide linker to the N-terminus of the complementary VH or VL domain of a second binding site; the C-terminus of the complementary VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a third peptide linker; and A second antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a fourth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0251] In an embodiment of the present disclosure, the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen. In one embodiment, the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen.

[0252] In one embodiment, the C-terminus of the second Fab heavy chain is fused via a second peptide linker to the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site specific for the second antigen. In one embodiment, the C-terminus of the second Fab heavy chain is fused via a second peptide linker to the N-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site specific for the second antigen.

[0253] In one embodiment, the C-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a third peptide linker. In one embodiment, the C-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a third peptide linker.

[0254] In one embodiment, the first antigen binding molecule consists of or comprises a first and a second polypeptide, wherein: a) the first polypeptide consists of or comprises the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. It consists of or includes:

[0255] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a third, fourth and fifth polypeptide, wherein: a) the third polypeptide consists of or comprises the light chain of a second Fab; b) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a second peptide linker; iii. either a complementary VH or VL domain of a second binding site specific for a second antigen; iv. a third peptide linker, and v. a first Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; consisting of or comprising: c) the third polypeptide comprises from its N-terminus to its C-terminus: i. a fourth peptide linker, and ii. a second Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus Includes.

[0256] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH and VL domains of the second binding molecule can be non-covalently linked to form a second binding site.

[0257] In one embodiment, the present disclosure relates to a set of antigen-binding molecules consisting of or comprising a first antigen-binding molecule having a structure as depicted in FIG. 1A and a second antigen-binding molecule having a structure as depicted in FIG. 1B.

[0258] Set of antigen-binding molecules in (1 1 / 2B027) format and (1 1 / 2)B038 Fc-KiH format In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first targeting moiety comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker, and iii. either the first VH or VL domain of a second binding site specific for a second antigen; A first antigen-binding molecule consisting of or comprising: a first antigen-binding molecule in which a first targeting moiety is fused to the N-terminus of either the VH or VL domain of the second binding site via a first peptide linker; b) from its N-terminus to its C-terminus: i. a second targeting moiety comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a second peptide linker; iii. an Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; iv. a third peptide linker, and v. A complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: a second targeting moiety is fused to the N-terminus of the first Fc region subunit via a second peptide linker; the C-terminus of the complementary VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a third peptide linker; and A second antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a fourth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0259] In one embodiment, the first targeting moiety is fused to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker. In one embodiment, the first targeting moiety is fused to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a third peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the VL domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a third peptide linker.

[0260] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from or comprises the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab.

[0261] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a)Below: i. a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. A first antigen-binding molecule consisting of or comprising: A first antigen-binding molecule in which the C-terminus of a first Fab heavy chain is fused to the N-terminus of either the VH or VL domain of a second binding site via a first peptide linker; b)Below: I. A second Fab comprising a third binding site specific for a third antigen or a third antigen epitope; II. A second peptide linker, III. An Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain; IV. A third peptide linker, and V. A complementary VH or VL domain of a second binding site specific for a second antigen In the second antigen-binding molecule, which consists of or comprises: the C-terminus of the second Fab heavy chain is fused to the N-terminus of the first Fc region subunit via a second peptide linker; the C-terminus of the complementary VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a third peptide linker; and A second antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a fourth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0262] In one embodiment, the C-terminus of the first Fab heavy chain is fused to the N-terminus of the VH domain of the first antigen binding molecule of the second binding site specific for the second antigen via a first peptide linker. In one embodiment, the C-terminus of the first Fab heavy chain is fused to the N-terminus of the VL domain of the first antigen binding molecule of the second binding site specific for the second antigen via a first peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the complementary VH domain of the second antigen binding molecule of the second binding site specific for the second antigen via a third peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the VL domain of the second antigen binding molecule of the second binding site specific for the second antigen via a third peptide linker.

[0263] In one embodiment, the first antigen binding molecule consists of or comprises a first and a second polypeptide, wherein: a) the first polypeptide consists of or comprises the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. It consists of:

[0264] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a third, fourth and fifth polypeptide, wherein: a) the third polypeptide consists of or comprises the light chain of a second Fab; b) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a second peptide linker; iii. a first Fc region subunit composed of, from its N-terminus to its C-terminus, a CH2 and a CH3 domain; iv. a third peptide linker, and v. either the VH or VL domain of a second binding site specific for a second antigen It consists of c) the fifth polypeptide has from its N-terminus to its C-terminus: iii. a fourth peptide linker, and iv. from its N-terminus to its C-terminus, a second Fc region subunit composed of a CH2 and a CH3 domain. Includes.

[0265] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH and VL domains of the second binding molecule can be non-covalently linked to form a second binding site.

[0266] In one embodiment, the present disclosure relates to a set of antigen-binding molecules consisting of or including a first antigen-binding molecule having a structure as depicted in FIG. 1A and a second antigen-binding molecule having a structure as depicted in FIG. 1C.

[0267] Set of antigen-binding molecules in (1 1 / 2)B027 format and (2 1 / 2)B064 Fc-KiH format In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first targeting moiety comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. A first antigen-binding molecule consisting of or comprising: a first antigen-binding molecule in which a first targeting moiety is fused to the N-terminus of either the VH or VL domain of the second binding site via a first peptide linker; b)Below: i. a second targeting moiety comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a third targeting moiety comprising a fourth binding site specific for a third antigen or a third antigen epitope; iii. a second peptide linker; iv. a fourth peptide linker; v. A first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. vi. a third peptide linker; vii. a complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: a second targeting moiety is fused to the N-terminus of the first Fc region subunit via a second peptide linker; and a third targeting moiety is fused to the N-terminus of the second Fc region subunit via a fourth peptide linker; and A second antigen-binding molecule, in which the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a third peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0268] In one embodiment, the first targeting moiety is fused to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker. In one embodiment, the first targeting moiety is fused to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen via a first peptide linker.

[0269] In one embodiment, the first Fc region subunit is fused to the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a third peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the VL domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a third peptide linker.

[0270] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab.

[0271] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a)Below: i. a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. A first antigen-binding molecule consisting of or comprising: a first antigen-binding molecule in which the C-terminus of a first Fab heavy chain is fused to the N-terminus of either the VH or VL domain of a second binding site via a first peptide linker; b)Below: i. a second Fab comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a third Fab comprising a fourth binding site specific for a third antigen or a third antigen epitope; iii. a second peptide linker; iv. a fourth peptide linker; v. A first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. vi. a third peptide linker; vii. a complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: the C-terminus of the second Fab heavy chain is fused to the N-terminus of the first Fc region subunit via a second peptide linker; the C-terminus of the third Fab heavy chain is fused to the N-terminus of the second Fc region subunit via a fourth peptide linker; and A second antigen-binding molecule, in which the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of the first Fc region subunit via a third peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0272] In one embodiment, the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of the VH domain of the first antigen-binding molecule of the second binding site specific for the second antigen. In one embodiment, the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of the VL domain of the first antigen-binding molecule of the second binding site specific for the second antigen.

[0273] In one embodiment, the first Fc region subunit is fused to the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a third peptide linker. In one embodiment, the first Fc region subunit is fused to the N-terminus of the VL domain of the second antigen-binding molecule of the second binding site specific for the second antigen via a third peptide linker.

[0274] In one embodiment, the targeting moiety is an antibody or antibody fragment. In one embodiment, the targeting moiety is selected from the group consisting of Fab, scFab, Fab', scFv, dsFv, and VHH. In one embodiment, the targeting moiety is a Fab.

[0275] In one embodiment, the first antigen binding molecule consists of or comprises a first and a second polypeptide, wherein: a) the first polypeptide consists of or comprises the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker, and iii. either the VH or VL domain of a second binding site specific for a second antigen. It consists of:

[0276] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a third, fourth, fifth and sixth polypeptide, wherein: a) the third polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a third Fab; ii. a fourth peptide linker; iii. a second Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; Including, b) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a second peptide linker; iii. a first Fc region subunit composed of, from its N-terminus to its C-terminus, a CH2 and a CH3 domain; iv. a third peptide linker; v. either the complementary VH or VL domain of the second binding site Including, c) the fifth polypeptide comprises a light chain of a second Fab, and d) the sixth polypeptide comprises the light chain of the third Fab.

[0277] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH and VL domains of the second binding molecule can be non-covalently linked to form a second binding site.

[0278] In one embodiment, the present disclosure relates to a set of antigen-binding molecules consisting of or including a first antigen-binding molecule having a structure as depicted in Figure 1A and a second antigen-binding molecule having a structure as depicted in Figure 4A. In one embodiment of the present disclosure, the set of antigen-binding molecules has a structure as depicted in Figure 4C.

[0279] Set of antigen-binding molecules in (1 1 / 2)B036 Fc-KiH format and (1 1 / 2)B038 Fc-KiH format In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first targeting moiety comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker; v. A first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. In the first antigen-binding molecule consisting of or comprising: a first targeting moiety is fused to the N-terminus of either the VH or VL domain of the second binding site region via a first peptide linker; the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker; and A first antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a third peptide linker; b) from its N-terminus to its C-terminus: i. a second targeting moiety comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a fourth peptide linker; iii. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; iv. a fifth peptide linker; v. A complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule consisting of or comprising: the C-terminus of the second targeting moiety is fused to the N-terminus of the third Fc region subunit via a fourth peptide linker; the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of a third Fc region subunit via a fifth peptide linker; and a second antigen-binding molecule, wherein the N-terminus of the fourth Fc region subunit is fused to a sixth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0280] In one embodiment, the first targeting moiety is fused to the N-terminus of the VH domain of the first antigen binding molecule of the second binding site via a first peptide linker. In one embodiment, the first targeting moiety is fused to the N-terminus of the VL domain of the first antigen binding molecule of the second binding site region via a first peptide linker.

[0281] In one embodiment, the C-terminus of the VH domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker. In one embodiment, the C-terminus of the VL domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker.

[0282] In one embodiment, the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site is fused to the C-terminus of the third Fc region subunit via a fifth peptide linker. In one embodiment, the N-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site is fused to the C-terminus of the third Fc region subunit via a fifth peptide linker.

[0283] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker; v. A first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. In the first antigen-binding molecule consisting of or comprising: the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of either the VH or VL domain of the second binding site; the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker; and A first antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a third peptide linker; b) from its N-terminus to its C-terminus: i. a second Fab comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a fourth peptide linker; iii. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; iv. a fifth peptide linker; v. A complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule consisting of or comprising: the C-terminus of the second Fab heavy chain is fused to the N-terminus of a third Fc region subunit via a fourth peptide linker; the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of a third Fc region subunit via a fifth peptide linker; and A second antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a sixth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0284] In one embodiment, the C-terminus of the first Fab heavy chain is fused to the N-terminus of the VH domain of the first antigen binding molecule of the second binding site via a first peptide linker. In one embodiment, the C-terminus of the first Fab heavy chain is fused to the N-terminus of the VL domain of the first antigen binding molecule of the second binding site region via a first peptide linker.

[0285] In one embodiment, the C-terminus of the VH domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker. In one embodiment, the C-terminus of the VL domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker.

[0286] In one embodiment, the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site is fused to the C-terminus of the third Fc region subunit via a fifth peptide linker. In one embodiment, the N-terminus of the complementary VL domain of the first antigen-binding molecule of the second binding site is fused to the C-terminus of the third Fc region subunit via a fifth peptide linker.

[0287] In one embodiment of the present disclosure, the first antigen-binding molecule consists of or comprises a first, second, and third polypeptide, wherein: a) the first polypeptide consists of or comprises the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker, and v. a first Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; Including, c) the third polypeptide comprises from its N-terminus to its C-terminus: i. a third peptide linker, and ii. a second Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus Includes.

[0288] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a fourth, fifth, and sixth polypeptide, wherein: a. the fourth polypeptide comprises the light chain of the second Fab; b. the fifth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a fourth peptide linker; iii. a third Fc region subunit composed of a CH2 and a CH3 domain from its N-terminus to its C-terminus; iv. a fifth peptide linker, and v. A complementary VH or VL domain of a second binding site specific for a second antigen. Including, c. the sixth polypeptide comprises from its N-terminus to its C-terminus: i. a sixth peptide linker, ii. from its N-terminus to its C-terminus, a fourth Fc region subunit composed of a CH2 and a CH3 domain. Includes.

[0289] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH and VL domains of the second binding molecule can be non-covalently linked to form a second binding site.

[0290] In one embodiment, the present disclosure relates to a set of antigen-binding molecules consisting of or including a first antigen-binding molecule having a structure as depicted in FIG. 1B and a second antigen-binding molecule having a structure as depicted in FIG. 1C.

[0291] Set of antigen-binding molecules in (1 1 / 2)B036 Fc-KiH format and (2 1 / 2)B064 Fc-KiH format In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first targeting moiety comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker, and v. A first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. In the first antigen-binding molecule, which consists of or comprises: a first targeting moiety is fused to the N-terminus of either the VH or VL domain of the second binding site via a first peptide linker; the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker; and A first antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a third peptide linker; b)Below: i. a second targeting moiety comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a third targeting moiety comprising a fourth binding site specific for a third antigen or a third antigen epitope; iii. a fourth peptide linker; iv. a sixth peptide linker; v. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; vi. a fifth peptide linker; vii. a complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: a second targeting moiety is fused to the N-terminus of the third Fc region subunit via a fourth peptide linker; a third targeting moiety is fused to the N-terminus of the fourth Fc region subunit via a sixth peptide linker; a second antigen-binding molecule, in which the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of a third Fc region subunit via a fifth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0292] In one embodiment, the first targeting moiety is fused to the N-terminus of the VH domain of the first antigen binding molecule of the second binding site via a first peptide linker. In one embodiment, the first targeting moiety is fused to the N-terminus of the VL domain of the first antigen binding molecule of the second binding site region via a first peptide linker.

[0293] In one embodiment, the C-terminus of the VH domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker. In one embodiment, the C-terminus of the VL domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker.

[0294] In one embodiment, the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site is fused to the C-terminus of the third Fc region subunit via a fifth peptide linker. In one embodiment, the N-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site is fused to the C-terminus of the third Fc region subunit via a fifth peptide linker.

[0295] In one embodiment, the present disclosure provides a method for producing a method for the preparation of a composition comprising: a) from its N-terminus to its C-terminus: i. a first Fab comprising a first binding site specific for a first antigen or a first antigen epitope; ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker; v. A first Fc region composed of a first and a second Fc region subunit, each Fc region subunit being composed of a CH2 and a CH3 domain. In the first antigen-binding molecule, which consists of or comprises: the C-terminus of the first Fab heavy chain is fused via a first peptide linker to the N-terminus of either the VH or VL domain of a second binding site; the C-terminus of either the VH or VL domain of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker; and A first antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a third peptide linker; b)Below: i. a second Fab comprising a third binding site specific for a third antigen or a third antigen epitope; ii. a third Fab comprising a fourth binding site specific for a third antigen or a third antigen epitope; iii. a fourth peptide linker; iv. a sixth peptide linker; v. a second Fc region composed of a third and a fourth Fc region subunit, each Fc region subunit composed of a CH2 and a CH3 domain; vi. a fifth peptide linker; vii. a complementary VH or VL domain of a second binding site specific for a second antigen. In the second antigen-binding molecule, which consists of or comprises: the C-terminus of the second Fab heavy chain is fused to the N-terminus of a third Fc region subunit via a fourth peptide linker; the C-terminus of the third Fab heavy chain is fused to the N-terminus of the fourth Fc region subunit via a sixth peptide linker; a second antigen-binding molecule, in which the N-terminus of the complementary VH or VL domain of the second binding site is fused to the C-terminus of a third Fc region subunit via a fifth peptide linker. The present invention relates to a set of antigen-binding molecules consisting of or comprising:

[0296] In one embodiment, the C-terminus of the first Fab heavy chain is fused to the N-terminus of the VH domain of the first antigen binding molecule of the second binding site via a first peptide linker. In one embodiment, the C-terminus of the first Fab heavy chain is fused to the N-terminus of the VL domain of the first antigen binding molecule of the second binding site region via a first peptide linker.

[0297] In one embodiment, the C-terminus of the VH domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker. In one embodiment, the C-terminus of the VL domain of the first antigen-binding molecule of the second binding site is fused to the N-terminus of the first Fc region subunit via a second peptide linker.

[0298] In one embodiment, the N-terminus of the complementary VH domain of the second antigen-binding molecule of the second binding site is fused to the C-terminus of the third Fc region subunit via a fifth peptide linker. In one embodiment, the N-terminus of the complementary VL domain of the second antigen-binding molecule of the second binding site is fused to the C-terminus of the third Fc region subunit via a fifth peptide linker.

[0299] In one embodiment of the present disclosure, the first antigen-binding molecule consists of or comprises a first, second, and third polypeptide, wherein: a) the first polypeptide consists of or comprises the light chain of a first Fab, b) the second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a first Fab, ii. a first peptide linker; iii. either the VH or VL domain of a second binding site specific for a second antigen; iv. a second peptide linker, and v. a first Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus; Including, c) the third polypeptide comprises from its N-terminus to its C-terminus: i. a third peptide linker, and ii. a second Fc region subunit consisting of a CH2 and a CH3 domain from its N-terminus to its C-terminus Includes.

[0300] In one embodiment of the disclosure, the second antigen binding molecule consists of or comprises a fourth, fifth, sixth and seventh polypeptide, wherein: a) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a third Fab; ii. a sixth peptide linker; iii. comprising from its N-terminus to its C-terminus a fourth Fc region subunit composed of a CH2 and a CH3 domain; b) the fifth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of a second Fab, ii. a fourth peptide linker; iii. a third Fc region subunit composed of a CH2 and a CH3 domain from its N-terminus to its C-terminus; iv. a fifth peptide linker; v. The complementary VH or VL domain of the second binding site Including, c) the sixth polypeptide comprises a light chain of a second Fab, and d) the seventh polypeptide comprises the light chain of the third Fab.

[0301] In one embodiment of the present disclosure, in the set of antigen-binding molecules according to the present disclosure, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond. In one embodiment of the present disclosure, the VH and VL domains of the second binding molecule can be non-covalently linked to form a second binding site.

[0302] In one embodiment, the present disclosure relates to a set of antigen-binding molecules consisting of or including a first antigen-binding molecule having a structure as depicted in Figure 1B and a second antigen-binding molecule having a structure as depicted in Figure 4A. In one embodiment of the present disclosure, the set of antigen-binding molecules has a structure as depicted in Figure 4D.

[0303] antibody The antibodies or antibody fragments, as well as the VH and VL domains, used in the antigen-binding molecules according to the present disclosure may be from any animal species, such as mouse, rat, human or non-human primate, and preferably are human in origin or may be obtained by a humanization approach.

[0304] Linker An antigen binding molecule according to the present disclosure can be designed such that its individual components (such as targeting moieties or unpaired VH and VL domains) are fused directly to each other or indirectly via a linker.

[0305] In certain embodiments, the individual components of the antigen-binding molecule according to the present disclosure are genetically fused to each other. Such fusion can be achieved by several strategies, including but not limited to peptide or polypeptide fusion between the N-terminus and C-terminus of the peptide or polypeptide, fusion via disulfide bonds, and fusion via chemical cross-linking reagents.

[0306] The composition and length of the linker can be determined according to methods known in the art, and they can be tested for effectiveness. Preferably, the linker is non-immunogenic. Non-immunogenic peptide linkers used herein can include glycine-alanine polymers, alanine-serine polymers, and other flexible peptide linkers. In one embodiment, the linker is a peptide linker. In one embodiment, the linker is a peptide linker that includes one or more amino acid residues linked by peptide bonds known in the art. The peptide linker should have a length suitable for fusing the two components so that they are in the correct configuration relative to each other so that they retain or acquire the desired activity or functionality.

[0307] The peptide linker may comprise the following amino acid residues: Gly, Ser, Ala, or Thr. A suitable non-immunogenic peptide linker is a glycine-serine polymer, e.g., (GS) n (SEQ ID NO: 36), (G4S) n (SEQ ID NO:37), (SG4) n (SEQ ID NO: 38), (GSGGS) n (SEQ ID NO:39), (GGGS) n (SEQ ID NO: 40) or G4 (SG4) n (SEQ ID NO:41), where n is an integer from 1 to 10, typically from 2 to 4. In one embodiment, the peptide linker is selected from the group consisting of, but not limited to, QPKAAP (SEQ ID NO:42), ASTKGP (SEQ ID NO:43), (G4S)3 (SEQ ID NO:44), (GGS)3 (SEQ ID NO:45), DKTHTCPPCP (SEQ ID NO:46), QPKAAPDKTHTCPPCP (SEQ ID NO:47), and ASTKGPDKTHTCPPCP (SEQ ID NO:48).

[0308] The peptide linker may also be derived from an immunoglobulin light or heavy chain constant domain, such as a CLκ or CLλ domain or a CH1 domain, but not all residues of such a constant domain, for example only the first 5-12 amino acid residues. In one embodiment, the peptide linker is not an immunoglobulin light or heavy chain constant domain. In one embodiment, the peptide linker is not a CLκ, CLλ, CH1, CH2 or CH3 domain. An exemplary peptide linker that may be used in the antigen binding molecule is derived from an immunoglobulin light or heavy chain constant domain and is QPKAAP (SEQ ID NO: 49) or ASTKGP (SEQ ID NO: 50).

[0309] 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 comprise one or more of its interchain cysteines. The presence of the interchain cysteines allows the formation of a dimeric peptide linker (or hinge region) by disulfide bridges in the situation where two such hinge peptide linkers are used. The presence of a dimeric peptide linker or hinge region further promotes and stabilizes the dimerization of two Fc region subunits that may be present in the antigen binding molecule according to the present disclosure. Exemplary peptide linkers derived from human IgG hinges suitable for dimerization are DKTHTCPPCP (SEQ ID NO: 46), KTHTCPPCP (SEQ ID NO: 32) or EPKSCDKTHTCPPCP (SEQ ID NO: 34).

[0310] In one embodiment, the peptide linkers present in a set of antigen-binding molecules according to the present disclosure are identical. In one embodiment, the peptide linkers are different. In one embodiment, the peptide linkers are the same length. In one embodiment, the peptide linkers are different lengths.

[0311] In one embodiment, the peptide linker according to the present disclosure is composed only of naturally occurring amino acid residues. In one embodiment, the peptide linker is composed only of naturally occurring amino acid residues, except for cysteine. In one embodiment, the peptide linker according to the present disclosure is composed of amino acid residues A, Q, D, P, H, G, S, E, T, K, and C. In one embodiment, the peptide linker of the antigen binding molecule according to the present disclosure has a length of at least 5 amino acid residues.

[0312] In one embodiment, the peptide linker of the antigen-binding molecule according to the present disclosure has a length of 5 to 50 amino acid residues, preferably 5 to 29 amino acid residues. In one embodiment, the peptide linker of the antigen-binding molecule according to the present disclosure has a length of 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 one embodiment, the peptide linker of the antigen-binding molecule according to the present disclosure has a length of 5 to 49 amino acid residues. In one embodiment, the peptide linker has a length of 5 to 40 amino acid residues. In one embodiment, the peptide linker has a length of 9 to 29 amino acid residues. In one embodiment, the peptide linker has a length of 5 to 29 amino acid residues. In one embodiment, the peptide linker of the antigen binding molecule according to the present disclosure has a length selected from the following: 5 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, and 49 amino acid residues.

[0313] In one embodiment, the peptide linker of the antigen binding molecule according to the present disclosure has a length of 5 amino acid residues. In one embodiment, the peptide linker has a length of 9 amino acid residues. In one embodiment, the peptide linker has a length of 10 amino acid residues. In one embodiment, the peptide linker has a length of 15 amino acid residues. In one embodiment, the peptide linker has a length of 19 amino acid residues. In one embodiment, the peptide linker has a length of 20 amino acid residues. In one embodiment, the peptide linker has a length of 25 amino acid residues. In one embodiment, the peptide linker has a length of 29 amino acid residues. In one embodiment, the peptide linker has a length of 40 amino acid residues. In one embodiment, the peptide linker has a length of 45 amino acid residues. In one embodiment, the peptide linker has a length of 49 amino acid residues.

[0314] In one embodiment, the peptide linker fusing a targeting moiety to the VH or VL of the second binding site of the antigen-binding molecule according to the present disclosure has a length of 5 to 40 amino acid residues, preferably 5 to 20 amino acid residues. In one embodiment, the peptide linker fusing a targeting moiety to the VH or VL of the second binding site of the antigen-binding molecule according to the present disclosure has a length of 5 to 40 amino acid residues. In one embodiment, the peptide linker fusing a targeting moiety to the VH or VL of the second binding site of the antigen-binding molecule according to the present disclosure does not include an IgG Fc region. In one embodiment, the peptide linker fusing a targeting moiety to the VH or VL of the second binding site of the antigen-binding molecule according to the present disclosure is composed only of naturally occurring amino acid residues. In one embodiment, the peptide linker is composed only of naturally occurring amino acid residues except C. In one embodiment, the peptide linker is composed of amino acid residues selected from the group of A, Q, D, P, H, G, S and E.

[0315] In one embodiment, the peptide linker for fusing a targeting moiety to the VH or VL of the second binding site of the antigen-binding molecule according to the present disclosure is selected from those having a length of 5, 10, 20 or 40 amino acid residues. In one embodiment, the peptide linker has a length of 5 amino acid residues. In one embodiment, the peptide linker has a length of 10 amino acid residues. In one embodiment, the peptide linker has a length of 20 amino acid residues. In one embodiment, the peptide linker has a length of 40 amino acid residues. In one embodiment, the peptide linker for fusing a targeting moiety to the VH or VL of the second binding site of the antigen-binding molecule according to the present invention has a length of 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 one embodiment, the peptide linker for fusing a targeting moiety to the VH or VL of the second binding site of the antigen-binding molecule according to the present invention has a length of 5 to 20 amino acid residues.

[0316] In one embodiment, the peptide linker that fuses the targeting moiety to the N-terminus of the VH or VL domain of the second binding site of the antigen-binding molecule of the present disclosure is selected from those having a length of 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, or 40 amino acid residues, preferably 5 to 20 amino acid residues, preferably 5 or 20 amino acid residues.

[0317] In one embodiment, the peptide linker is selected from those having a length of 5 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 10 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 20 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 40 amino acid residues.

[0318] In one embodiment, the peptide linker fusing a targeting moiety to the VH or VL of the second binding site of the antigen-binding molecule according to the present disclosure has a length of 5 to 45 amino acid residues. In one embodiment, the peptide linker has a length of 10 to 45 amino acid residues. In one embodiment, the peptide linker has a length of 10 to 45 amino acid residues, preferably 10 to 25 amino acid residues. In one embodiment, the peptide linker has a length of 10 amino acid residues. In one embodiment, the peptide linker has a length of 15 amino acid residues. In one embodiment, the peptide linker has a length of 25 amino acid residues. In one embodiment, the peptide linker has a length of 45 amino acid residues. In one embodiment, the peptide linker has a length of 10, 15, 25 or 45 amino acid residues.

[0319] In one embodiment, the peptide linker fusing the targeting moiety to the VH or VL of the second binding site of the antigen binding molecule according to the present disclosure is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105.

[0320] In one embodiment, the peptide linker that fuses an Fc domain subunit to VH or VL of the second binding site of the antigen-binding molecule according to the present disclosure has a length of 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, or 5 to 25 amino acid residues.

[0321] In one embodiment, the peptide linker that fuses an Fc region subunit to the VH or VL of the second binding site of the antigen-binding molecule according to the present disclosure has a length of 20 to 50 amino acid residues, 20 to 45 amino acid residues, 20 to 40 amino acid residues, 20 to 35 amino acid residues, 20 to 30 amino acid residues, or 20 to 25 amino acid residues. In one embodiment, the peptide linker that fuses an Fc region subunit to the VH or VL of the second binding site of the antigen-binding molecule according to the present disclosure has a length of 5 to 29 amino acid residues.

[0322] In one embodiment, the peptide linker fusing the Fc domain subunit to the VH or VL of the second binding site of the antigen binding molecule of the present disclosure is selected from those having a length of 5, 9, 10, 20, 29, 40 or 49 amino acid residues.

[0323] In one embodiment, the peptide linker fusing the Fc domain subunit to the VH or VL of the second binding site of the antigen binding molecule according to the present disclosure has a length of 5, 9, 10, 20, 29, 40 or 49 amino acid residues.

[0324] In one embodiment, the peptide linker fusing the N-terminus of the Fc region subunit to the C-terminus of the VH or VL domain of the second binding site of the antigen-binding molecule according to the present disclosure has a length of 9 to 49 amino acid residues.

[0325] In one embodiment, the peptide linker fusing the N-terminus of the Fc region subunit to the C-terminus of the VH or VL domain of the second binding site of the antigen binding molecule of the present disclosure has a length of 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, or 49 amino acid residues. In one embodiment, the peptide linker fusing the N-terminus of the Fc region subunit to the C-terminus of the VH or VL domain of the second binding site of the antigen binding molecule of the present disclosure is selected from those having a length of 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, or 49 amino acid residues, preferably 29 or 49 amino acid residues.

[0326] In one embodiment, the peptide linker is selected from those having a length of 20 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 29 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 40 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 49 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 9 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 29 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 49 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 9 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 49 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of only naturally occurring amino acid residues. In one embodiment, the peptide linker is selected from those having a length of A, Q, D, P, H, G, S, E, T, K, and C.

[0327] In one embodiment, the peptide linker fusing the N-terminus of the Fc region subunit to the C-terminus of the VH or VL domain of the second binding site of the antigen binding molecule of the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:106, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:46.

[0328] In one embodiment, the peptide linker that fuses a targeting moiety to the N-terminus of the Fc region subunit of the antigen-binding molecule according to the present disclosure has a length of 5 to 20 amino acid residues. In one embodiment, the peptide linker has a length of 9 to 15 amino acid residues. In one embodiment, the peptide linker has an amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 46, and SEQ ID NO: 32.

[0329] In one embodiment, the peptide linker fusing the targeting moiety to the N-terminus of the Fc region subunit of the antigen binding molecule of the present disclosure is selected from those having a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues, preferably 15 amino acid residues. In one embodiment, the peptide linker is selected from those having a length of 15 amino acid residues. In one embodiment, the peptide linker has a length of 15 amino acid residues. In one embodiment, the peptide linker is composed of amino acid residues selected from the group of E, P, K, S, C, D, T, and H.

[0330] In one embodiment, the peptide linker fusing the C-terminus of the Fc region subunit to the N-terminus of the VH or VL domain of the second binding site of the antigen binding molecule according to the present disclosure is selected from or has a length of 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, or 40 amino acid residues, preferably 20 amino acid residues. In one embodiment, the peptide linker is selected from or has a length of 20 amino acid residues. In one embodiment, the peptide linker has a length of 20 amino acid residues.

[0331] In one embodiment, the peptide linker that fuses the C-terminus of the Fc region subunit to the N-terminus of the VH or VL domain of the second binding site of the antigen-binding molecule according to the present disclosure is selected from those having a length of 5 to 40 amino acid residues, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acid residues, or has that length. In one embodiment, the peptide linker has a length of 5 to 20 amino acid residues.

[0332] In one embodiment, preferred peptide linkers for use in antigen binding molecules according to the present disclosure are the following: GQPSG (SEQ ID NO: 35), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16), AQPAAPAPAE (SEQ ID NO: 51), AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 31), AQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 33), AQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 52), DQPAAPAPDAHEAPAPAH EAPAPAQGS (SEQ ID NO: 53), DQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 54), DQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 55), DQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 56), KTHT (SEQ ID NO: 107), KTHTCPPCP (SEQ ID NO: 32), and EPKSCDKTHTCPPCP (SEQ ID NO: 34).

[0333] In one embodiment, the peptide linker fusing the targeting moiety to either the VH or VL domain of the second binding site is selected from the group consisting of: GQPSG (SEQ ID NO: 35), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16), AQPAAPAPAE (SEQ ID NO: 51), AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 31) and AQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 52).

[0334] In one embodiment, the peptide linker fusing the targeting moiety to either the VH or VL domain of the second binding site is selected from the group consisting of the following: SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106. In one embodiment, the peptide linker fusing the C-terminus of the VH or VL domain of the second binding site to the N-terminus of the Fc region subunit is selected from the group consisting of the following: KTHTCPPCP (SEQ ID NO: 32), AQPAAPAPAEKTHTCPPCP (SEQ ID NO: 106), AQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 33), DQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 54), and DQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 56).

[0335] In one embodiment, the peptide linker fusing the N-terminus of the VH or VL domain of the second binding site to the C-terminus of the Fc region subunit is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16). In one embodiment, the peptide linker fusing the targeting moiety to the Fc region subunit is EPKSCDKTHTCPPCP (SEQ ID NO: 34). In one embodiment, the peptide linker fused to the N-terminus of the Fc region subunit is KTHTCPPCP (SEQ ID NO: 32). In one embodiment, the peptide linker present at the N-terminus of the Fc region subunit is KTHTCPPCP (SEQ ID NO: 32).

[0336] It is understood that the peptide linker used herein is not limited to only one of the above exemplified peptide linkers, but may include any combination of two or more such linkers fused together. For example, the peptide linker used herein may be constructed from a glycine-serine polymer and a sequence derived from an immunoglobulin hinge, and may further include the entire IgG Fc region.

[0337] Spacer The potency and efficacy of a set of antigen binding molecules disclosed herein in mediating killing of a target cell may vary depending, inter alia, on the target antigen or target antigen epitope (e.g., their distance from each other and / or to the cell surface, etc.) and the distance from their targeting moieties to their unpaired VH or VL domains, or the distance from each of the targeting moieties to the newly formed Fv domain of the on-cell forming triabody.

[0338] Thus, the efficacy of such a set of antigen-binding molecules in mediating target cell killing can be optimized by varying the distance between each targeting moiety and its unpaired VH or VL domain, which can be achieved by appropriately selecting the length of the spacer linking the targeting moiety to the unpaired VH or VL domain of the antigen-binding molecules disclosed herein.

[0339] In this sense, the length of each spacer of the antigen-binding molecules present in the set of antigen-binding molecules according to the present disclosure is selected to allow optimal non-covalent binding of the unpaired VH and VL domains of the two antigen-binding molecules, thereby allowing the formation of a functional antibody Fv domain when the two antigen-binding molecules are bound to their target antigens or antigen epitopes on target cells.

[0340] As used herein, "spacer" refers to any proteinaceous moiety, such as a peptide or polypeptide, comprising one or more amino acid residues linked by peptide bonds known in the art, which links a targeting moiety to an unpaired VH or VL domain of an antigen-binding molecule according to the present disclosure, as long as the antigen-binding molecule does not itself promote dimerization of the antigen-binding molecules present in the set of antigen-binding molecules. Such a spacer may contain, encompass, comprise, or consist of any of the peptide linkers disclosed herein, as well as combinations thereof. A suitable spacer for use in linking a targeting moiety to an unpaired VH or VL domain of an antigen-binding molecule according to the present disclosure may be any spacer used in the art to link peptides and / or proteins. Some suitable spacers include, for example, polypeptide spacers, such as, but not limited to, glycine spacers, serine spacers, mixed glycine / serine spacers, glycine and serine-rich spacers, spacers composed mostly of polar polypeptide fragments, or spacers comprising amino acid sequences that form random coil structures. Suitable spacers may further comprise constant domains of immunoglobulins, such as the CH1, CH2 or CH3 domains, as well as fragments or portions or combinations thereof. Suitable spacer regions may also comprise the entire IgG Fc region, which is composed of a pair of IgG region subunits of IgG, or other proteinaceous half-life extending moieties, such as serum albumin.

[0341] Accordingly, the present disclosure provides: a) from its N-terminus to its C-terminus: i. a first targeting moiety specific for a first antigen or a first antigen epitope; ii. a first peptide linker; iii. a first spacer; iv. either the VH or VL domain of an antibody Fv domain specific for CD3; v. optionally, a third peptide linker, and vi. Optionally, an Fc region A first antigen-binding molecule consisting of or comprising: b) from its N-terminus to its C-terminus: i. a second targeting moiety specific for a second antigen or a second antigen epitope; ii. a second peptide linker; iii. a second spacer; and iv. a complementary VH or VL domain of an antibody Fv domain specific for CD3; v. optionally, a third peptide linker, and vi. Optionally, an Fc region A second antigen-binding molecule consisting of or comprising: providing a set of antigen-binding molecules consisting of or comprising: wherein the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond, The first antigen or first antigen epitope and the second antigen or second antigen epitope are present on the same cell, and the lengths of the first and second spacers are selected based on the distance from the first antigen or first antigen epitope to the second antigen or second antigen epitope and / or based on the distance from the first antigen epitope to the cell surface and the distance from the second antigen epitope to the cell surface.

[0342] In one embodiment, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: a) from its N-terminus to its C-terminus: i. a first targeting moiety specific for a first antigen or a first antigen epitope; ii. a first spacer; iii. either the VH or VL domain of an antibody Fv domain specific for CD3; and iv.Optionally, Fc region A first antigen-binding molecule consisting of or comprising: b) from its N-terminus to its C-terminus: i. a second targeting moiety specific for a second antigen or a second antigen epitope; ii. a second spacer; and iii. a complementary VH or VL domain of an antibody Fv domain specific for CD3; iv.Optionally, Fc region A second antigen-binding molecule consisting of or comprising: providing a set of antigen-binding molecules consisting of or comprising: Here, the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond, the first antigen or the first antigen epitope and the second antigen or the second antigen epitope are present on the same cell, and the lengths of the first and second spacers are selected based on the distance from the first antigen or the first antigen epitope to the second antigen or the second antigen epitope and / or based on both the distance from the first antigen epitope to the cell surface and the distance from the second antigen epitope to the cell surface.

[0343] In one embodiment, the second antigen binding molecule further comprises or optionally further comprises a third targeting moiety specific for the second antigen or the second antigen epitope, optionally fused to the N-terminus of the second spacer via a peptide linker. In one embodiment, the first, second, third and / or fourth targeting moiety is a Fab or scFv.

[0344] In one embodiment, the C-terminus of the Fab heavy chain is fused to the N-terminus of the first or second spacer, or to the first or second peptide linker. In one embodiment, the first antigen or first antigen epitope and the second antigen or second antigen epitope are the same. In one embodiment, the first antigen or first antigen epitope and the second antigen or second antigen epitope are different. In one embodiment, the first antigen epitope and the second antigen epitope are present on the same antigen. In one embodiment, the first antigen epitope and the second antigen epitope are present on different antigens. In one embodiment, the first and second antigens or antigen epitopes are present on the same cell. In one embodiment, the first and second antigens are tumor associated antigens. In one embodiment, the cell is a tumor cell.

[0345] In one embodiment of the present disclosure, the length of the first and second spacers is selected based on the distance from the first antigen epitope to the second antigen epitope. In one embodiment, the length of the first and / or second spacer corresponds to the distance from the first antigen epitope to the second antigen epitope. In one embodiment, the sum of the lengths of the first and second spacers corresponds to the distance from the first antigen epitope to the second antigen epitope.

[0346] In one embodiment, the distance from the first antigenic epitope to the second antigenic epitope is about 15 Å, 20 Å, 30 Å, 40 Å, 50 Å, 60 Å, 65 Å, 70 Å, 75 Å, 80 Å, 85 Å, 90 Å, 95 Å, 100 Å, 110 Å, 120 Å, 130 Å, 140 Å, 150 Å, 160 Å, 170 Å, 180 Å, 190 Å, 200 Å, 225 Å, 250 Å, 275 Å, 300 Å, 350 Å, 400 Å, 450 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, or about 1000 Å.

[0347] As used herein, 1 angstrom [Å] corresponds to approximately 0.1 nanometer [nm].

[0348] For example, if the distance from the first antigen epitope to the second antigen epitope is about 100 Å, the first spacer and the second spacer can each be selected to have a length of 50 Å. Alternatively, the first spacer can be selected to have a length of 30 Å, followed by a second spacer to have a length of 70 Å, or vice versa.

[0349] However, for example, when the first antigen-binding molecule targets a more cell surface proximal epitope and the second antigen-binding molecule targets a more cell surface distal epitope, it is preferable to keep the spacer of the second antigen-binding molecule to a minimum or as short as possible. For example, when the distance from the first antigen epitope to the second antigen epitope is about 100 Å and the second antigen-binding molecule targets a more cell surface distal epitope, it is preferable to select a first spacer with a length of about 83 Å and a second spacer with a length of about 17 Å. In this scenario, the first spacer will act as a stalk by placing the unpaired variable domain at the same level as or in close proximity to the second antigen epitope. The presence of a short or minimal second spacer ensures sufficient flexibility to allow binding and functional complementation of the VH and VL domains. Such a minimal spacer is selected to have a length of about 5 to 20 amino acid residues, preferably 5 amino acid residues. Alternatively, such a minimum spacer is selected to have a length between about 17.5 Å and 70 Å, preferably about 17.5 Å.

[0350] The length of a peptide linker or spacer as used herein may be described as a number of amino acid residues, such as, for example, 5, 6, or 7 amino acid residues. Alternatively, the length of a peptide linker or spacer as used herein may be expressed in angstroms [Å], where the theoretical distance between two amino acid residues linked by a peptide bond corresponds to about 3.5 Å. Thus, the theoretical maximum length of an unstructured or linear peptide linker of 5 amino acid residues corresponds to about 17.5 Å, a linear peptide linker of 10 amino acid residues corresponds to about 35 Å, 20 amino acid residues corresponds to about 70 Å, and 40 amino acid residues corresponds to about 140 Å.

[0351] In another embodiment, the length of an IgG Fc region is about 65 Å. In one embodiment, the length of an antibody Fab fragment is about 60 Å. In one embodiment, the length of an antibody Fv domain is about 30 Å. Such lengths can also be considered when selecting an appropriate length for a spacer according to the present disclosure.

[0352] In one embodiment, in the set of antigen-binding molecules according to the present disclosure, the total length of the first spacer of the first antigen-binding molecule and the length of the second spacer of the second antigen-binding molecule is selected from the following: about 15 Å, 20 Å, 30 Å, 40 Å, 50 Å, 60 Å, 65 Å, 70 Å, 75 Å, 80 Å, 85 Å, 90 Å, 95 Å, 100 Å, 110 Å, 120 Å, 130 Å, 140 Å, 150 Å, 160 Å, 170 Å, 180 Å, 190 Å, 200 Å, 225 Å, 250 Å, 275 Å, 300 Å, 350 Å, 400 Å, 450 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, or about 1000 Å, preferably 15 Å to 200 Å.

[0353] In one embodiment, the length of the first spacer of the first antigen-binding molecule or the length of the second spacer of the second antigen-binding molecule according to the present disclosure is selected from those having a length of about 15 Å, 20 Å, 30 Å, 40 Å, 50 Å, 60 Å, 65 Å, 70 Å, 75 Å, 80 Å, 85 Å, 90 Å, 95 Å, 100 Å, 110 Å, 120 Å, 130 Å, 140 Å, 150 Å, 160 Å, 170 Å, 180 Å, 190 Å, or about 200 Å.

[0354] In one embodiment, the length of the first spacer of the first antigen-binding molecule and the length of the second spacer of the second antigen-binding molecule in the set of antigen-binding molecules according to the present disclosure are selected based on the difference between the distance from the first antigen epitope to the cell surface and the distance from the second antigen epitope to the cell surface.

[0355] In one embodiment, the sum of the lengths of the first and second spacers corresponds to the difference between the distance from the first antigenic epitope to the cell surface and the distance from the second antigenic epitope to the cell surface.

[0356] In one embodiment, the distance from the first antigenic epitope to the cell surface is about 10 Å, 15 Å, 20 Å, 30 Å, 40 Å, 50 Å, 60 Å, 65 Å, 70 Å, 75 Å, 80 Å, 85 Å, 90 Å, 95 Å, 100 Å, 110 Å, 120 Å, 130 Å, 140 Å, 150 Å, 160 Å, 170 Å, 180 Å, 190 Å, 200 Å, 225 Å, 250 Å, 275 Å, 300 Å, 350 Å, 400 Å, 450 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, 1000 Å.

[0357] In one embodiment, the distance from the second antigenic epitope to the cell surface is about 10 Å, 15 Å, 20 Å, 30 Å, 40 Å, 50 Å, 60 Å, 65 Å, 70 Å, 75 Å, 80 Å, 85 Å, 90 Å, 95 Å, 100 Å, 110 Å, 120 Å, 130 Å, 140 Å, 150 Å, 160 Å, 170 Å, 180 Å, 190 Å, 200 Å, 225 Å, 250 Å, 275 Å, 300 Å, 350 Å, 400 Å, 450 Å, 500 Å, 600 Å, 700 Å, 800 Å, 900 Å, or about 1000 Å.

[0358] In one embodiment, the distance from the first antigen epitope to the cell surface is equal to the distance from the second antigen epitope to the cell surface. "Equal" as used in this context means that the difference in distance is 15 Å or less. In one embodiment, the distance from the first antigen epitope to the cell surface is different from the distance from the second antigen epitope to the cell surface. "Different" as used in this context means that the difference in distance is more than 15 Å.

[0359] In one embodiment, the distance from the first antigen epitope to the cell surface is shorter than the distance from the second antigen epitope to the cell surface. As used in this context, "shorter" means that the first antigen epitope is at least 15 Å closer to the cell surface than the second antigen epitope. In one embodiment, the distance from the second antigen epitope to the cell surface is shorter than the distance from the first antigen epitope to the cell surface. As used in this context, "shorter" means that the second antigen epitope is at least 15 Å closer to the cell surface than the second antigen epitope.

[0360] In one embodiment, the distance from the first antigenic epitope to the cell surface is greater than the distance from the second antigenic epitope to the cell surface. "Greater" as used in this context means that the first antigenic epitope is at least 15 Å further from the cell surface than the second antigenic epitope.

[0361] In one embodiment, the distance from the second antigenic epitope to the cell surface is greater than the distance from the first antigenic epitope to the cell surface. "Larger" as used in this context means that the second antigenic epitope is at least 15 Å further from the cell surface than the first antigenic epitope.

[0362] In one embodiment, the first antigen epitope is a cell surface proximal epitope, and the second antigen epitope is a cell surface distal epitope. In one embodiment, the first antigen epitope is a cell surface distal epitope, and the second antigen epitope is a cell surface proximal epitope. As used herein, "cell surface proximal epitope" refers to an antigen epitope that is 15 Å or less away from the cell surface. As used herein, "cell surface distal epitope" refers to an antigen epitope that is 30 Å or more away from the cell surface.

[0363] In one embodiment, in the set of antigen binding molecules of the present disclosure, the first antigen binding molecule binds to a first antigen epitope at the cell surface proximal side, and the second antigen binding molecule binds to a second antigen epitope at the cell surface distal side. In another embodiment, the first antigen binding molecule binds to a first antigen epitope at the cell surface distal side, and the second antigen binding molecule binds to a second antigen epitope at the cell surface proximal side.

[0364] In one embodiment, a first antigen binding molecule binds to a first antigen epitope and a second antigen binding molecule binds to a second antigen epitope, wherein the distances of the first and second antigen epitopes to the cell surface are approximately equal.

[0365] In one embodiment, in a set of antigen-binding molecules according to the present disclosure, when the distance from a first antigen epitope to a cell surface is shorter than the distance from a second antigen epitope to a cell surface, the length of the first spacer of the first antigen-binding molecule is selected to be longer than the length of the second spacer of the second antigen-binding molecule.

[0366] In one embodiment, when the distance from the first antigen epitope to the cell surface is shorter than the distance from the second antigen epitope to the cell surface, the length of the first spacer of the first antigen-binding molecule is selected so as to correspond to the absolute value of the calculated difference between the distance from the first antigen epitope to the cell membrane and the distance from the second antigen epitope to the cell surface. In such an embodiment, the second spacer of the second antigen-binding molecule is absent or is selected to have a length of 5 to 20 amino acid residues, preferably 5 amino acid residues.

[0367] In one embodiment, in a set of antigen-binding molecules according to the present disclosure, when the distance from a first antigen epitope to a cell surface is greater than the distance from a second antigen epitope to a cell surface, the length of the first spacer of the first antigen-binding molecule is selected to be shorter than the length of the second spacer of the second antigen-binding molecule.

[0368] In one embodiment, when the distance from the first antigen epitope to the cell surface is greater than the distance from the second antigen epitope to the cell surface, the length of the second spacer of the second antigen-binding molecule is selected to correspond to the calculated difference between the distance from the first antigen epitope to the cell surface and the distance from the second antigen epitope to the cell surface. In such an embodiment, the first spacer of the first antigen-binding molecule is selected to be absent or to have a length of 5 to 20 amino acid residues, preferably 5 amino acid residues.

[0369] In one embodiment, when the distance from the first antigen epitope to the cell surface is equal to the distance from the second antigen epitope to the cell surface, the first spacer and the second spacer are both absent, or each has a length of 5 to 20 amino acid residues.

[0370] In one embodiment, the distance from the first and second antigenic epitopes to the cell surface is determined using computer-assisted modeling of the 3D structure of the extracellular domain of the antigen complexed with a targeting moiety.

[0371] In one embodiment, the present disclosure provides a method for selecting a spacer length to be used in an antigen binding molecule in a set of antigen binding molecules according to the present disclosure, wherein the method comprises the steps of: a) determining the distance from a first antigenic epitope to a cell surface; b) determining the distance from the second antigenic epitope to the cell surface; c) subtracting the distance determined in step a) from the distance determined in step b); d) if the absolute value calculated from step c) is 15 Å or more, selecting the length of the spacer of the antigen-binding molecule that binds to an antigen epitope closer to the cell surface and not selecting the peptide spacer of the antigen-binding molecule that targets an antigen epitope farther from the cell surface according to the absolute value calculated from step c); or If the absolute value calculated from step c) is 15 Å or less, the first and second spacers of the first and second antigen-binding molecules are not selected.

[0372] In one embodiment, the absolute value of the calculated difference between the distance from the first antigenic epitope to the cell membrane and the distance from the second antigenic epitope to the cell surface is about 15 Å, 20 Å, 30 Å, 40 Å, 50 Å, 60 Å, 65 Å, 70 Å, 75 Å, 80 Å, 85 Å, 90 Å, 95 Å, 100 Å, 110 Å, 120 Å, 130 Å, 140 Å, 150 Å, 160 Å, 170 Å, 180 Å, 190 Å, 200 Å, 225 Å, 250 Å, 275 Å, 300 Å, 350 Å, 400 Å, 450 Å, 500 Å, 600 Å, 700 Å, 800 Å, or about 900 Å.

[0373] The relative position and / or distance of the antigen epitope to the cell surface can be determined by various methods known in the art, such as computer-aided modeling of the three-dimensional structure of the extracellular domain of the antigen complexed with the targeting moiety. The three-dimensional structure of the protein / antigen of interest can be retrieved from publicly available sources, such as the Research Collaboratory for Structural Bioinformatics Protein Data Base (https: / / www.rcsb.org / ). Such 3D structures can be analyzed by molecular visualization software, such as PyMOL.

[0374] Alternatively, de novo modeling of 3D protein structures by X-ray protein crystallography, with or without complexation with a target site such as an antibody Fab fragment, may be used. The latter co-crystallization technique allows the exact location of the targeted epitope on the antigen of interest to be determined. However, co-crystallization is not applicable in all cases. Therefore, alternative approaches for epitope mapping can be applied, which are known in the art, such as array-based oligopeptide scanning, site-directed mutagenesis mapping, high-throughput shotgun mutagenesis epitope mapping, hydrogen-deuterium exchange (HDX), cross-linking mass spectrometry, etc. The epitopes thus determined can be mapped onto the 3D structural model of the protein of interest obtained from X-ray protein crystallography, and the desired distances can be determined by computational techniques as described above.

[0375] target antigen Individual antigen-binding molecules according to the present disclosure are suitable for targeting different antigens. Thus, sets or combinations of antigen-binding molecules according to the present disclosure are particularly suitable for targeting different antigens and / or antigen epitopes present on the same target cell.

[0376] The ability of an antigen-binding molecule according to the present disclosure to specifically bind to a target antigen can be measured by either enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance techniques (analyzed on a BIACORE T100 system) (Liljebladet al Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0377] Competitive assays can be used to identify antibodies, antibody fragments, antigen-binding molecules, or targeting moieties that cross-compete with a reference antibody for binding to a particular antigen or epitope.

[0378] Thus, the targeting moiety of the antigen-binding molecule according to the present disclosure targets one antigen or antigen epitope. However, in a set of antigen-binding molecules according to the present disclosure, each of the two targeting moieties preferably targets a different antigen via their targeting moiety. The two antigens are selectively expressed on the surface of one cell, such as a cancer cell or tumor cell. When both antigen-binding molecules in a set of antigen-binding molecules according to the present disclosure bind to their respective antigens or antigen epitopes via their targeting moieties, the unpaired VL and VH domains come into close proximity and interact with each other to reconstitute the original antibody Fv domain. Thus, the trispecific heteromeric antibody molecule thus formed on-cell or on-target can target three different antigens, respectively, monovalently or bivalently. The ability to monovalently target three different antigens is a particularly useful feature of the set of antigen-binding molecules according to the present disclosure.

[0379] In one embodiment, the first and / or second antigens bound by the targeting moiety of the antigen binding molecule of the present disclosure are antigens associated with a pathological condition, such as antigens presented on tumor cells, virus-infected cells, or antigens expressed at inflammatory sites. Other suitable antigens include cell surface antigens (such as cell surface receptors), free antigens in serum, and / or antigens in the extracellular matrix. Preferably, such antigens are tumor-associated antigens.

[0380] In one embodiment, the antigen is a human antigen. In one embodiment, the first and / or second antigen is a tumor-associated antigen, in particular an antigen presented on tumor cells or cells of the tumor stroma. In one embodiment, the first antigen is an HLA-restricted peptide. In one embodiment, the first antigen is a peptide / HLA-A0201 complex.

[0381] Non-limiting examples of (tumor-associated) antigens include, for example, AR, AGR2, A1G1, AKAP1, AKAP2, ANGPT1, ANGPT2, ANPEP, ANGPTL3, APOC1, ANGPTL4, AITGAV, AZGP1, BMP6, BRCA1, BAD, BAG1, BCL2, BL6R, BA2, BPAG1, CDK2, CD52, CD20, CD19, CD4, CD8, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, CDK3, CDK4. , CDK5, CDK6, CDK7, CDK9, CLDN3, CLN3, CYB5, CYC1, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6, CXCL9, CHGB, CDH20, CDH7, CDH8, CDH9, CD44, CDH1, CDH1 0, CDH19, CDH20, CDH7, CDH9, CDH13, CDH18, CDH19, CANT1, CAV1, CDH12, CD164, COL6A1, CCL2, CDH5, COL18A1, CHGA, CHGB, CLU, COL1A1, COL6A1, CCNA1, C CNA2, CCND1, CCNE1, CCNE2, COL6A1, CTNNB1, CTSB, CLDN7, CLU, CD44APC, COL4A3, DSfHA, DAB2JP, DES, DNCL1, DD2, DL2, EL24, EGF, E2F1, EGFR, ENO1, ER BB2, ESR1, ESR2, EL2, EStHA, ELAC2, ENO2, ENO3, ERBB2, ESR1, ESR2, EDG1, EFNA1, EFNA3, EFNB2, EPHB4, ESR1, ESR2, EGF, ERK8, EL12A, EL1A, EL24, ENΗA, ELK, ECGF1, EREG, EDG1, ENG, E-cadherin, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FASN, FLJ12584, FLJ25530, F1GF, FLT1, FGFR3, F3, FOSL1, FLRT1, IL12A, IL1A, IL1B, IL2, INHA, IGF1, IGF2, IL12A,<h2 style=";text-align:left;direction:ltr">IL1A, IL1B, IL2, INHA, IGF1R, IL2, IGFBP6, IL1A, IL1B, IGFBP3, IGFBP6, INSL4, IL6ST, ITG6, IGF1, IGF2, INSL3, INSL4, IFNA1, IFNB1, IFNG, IL1B, IL6 、IGFBP2、IL2RA、IL6、IGF1、IGF2、IGFBP3、IGFBP6、ITGA1、IGF1、ITGA6、ITG B4、INSL3、INSL4、IL29、IL8、ITGB3、GRP、GNRH1、GAGEB1、GAGEC1、GGT1、GSTP 1、GATA3、GABRP、GNAS1、GSN、H1P1、HUMCYT2A、HGF、JAG1、JUN、LAMA5、S100A 2、SCGB1D2、SCGB2A1、SCGB2A2、SPRR1B、SHBG、SERP1NA3、SHBG、SLC2A2、SLC3 3A1、SLC43A1、STEAP、STEAP2、SERP1NF1、SERPINB5、SERPINE1、STAB1、TGFA 、TGFB1、TGFB2、TGFB3、TNF、TNFSF10、TGFB1I1、TP53、TPM1、TPM2、TRPC6、TGF A、THBS、TEE、TNFRSF6、TNFSF6、TOP2A、TP53、THBS1、THBS2、THBS4、TNFAIP2 、TP53、TEK、TGFA、TGFB1、TGFB2、TGFBR1、TGFA、TEV1P3、TGFB3、TNFA1P2、1TG B3、THBS1、THBS2、VEGF、VEGFC、ODZ1、PAWR、PLG、PAP、PCNA、PRKCQ、PRKD1、P RL、PECAM1、PF4、PROK2、PRL、PAP、PLAU、PRL、PSAP、PART1、PATE、PCA3、P1AS2 、PGF、PGR、PLAU、PGR、PLXDCl、PTEN、PTGS2、PDGF、MYC、MMP2、MMP9、MSMB、MA CMARCKS、MT3、MUC1、MAP2K7、MKi67、MTSS1、M1B1、MDK、NOX5、NR6A1、NR1H3、N R1I3、NR2F6、NR4A3、NR1H2、NR1H4、NR1I2、NR2C1、NR2C2、NR2E1、NR2E3、NR2 F1、NR2F2、NR3C1、NR3C2、NR4A1、NR4A2、NR5A1、NR5A2、NR6A1、NROB1、NROB2、NR1D2, NR1D1, NTN4, NRP1, NRP2, NGFB, NGFR, NME1, KLK6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, K6HF, KA2, KRT2A, KLK6, KLK3, KRT1, KDR, KLK5, KRT19, KLF5, KRT19, KRTHB6, RARB, RAC2, and ROBO2.

[0382] In one embodiment, the targeting moiety binds to a tumor associated antigen. In one embodiment, the first and second targeting moieties bind to a tumor associated antigen. In one embodiment, the first, second, third and / or fourth targeting moieties bind to a tumor associated antigen. In one embodiment, the first, second, third and / or fourth targeting moieties bind to a tumor associated antigen. In one embodiment, the first, second, third and / or fourth targeting moieties bind to a first and second tumor associated antigen.

[0383] In one embodiment, the first, second, third and / or fourth targeting moiety is a Fab or scFv. In one embodiment, the first, second, third and / or fourth targeting moiety is a Fab. In one embodiment, the first and / or second antigen is a first and / or second tumor associated antigen. In one embodiment, the first and / or third antigen is a tumor associated antigen. In one embodiment, the first and / or second antigen is a tumor associated antigen. In one embodiment, the first and second Fab specifically bind to a tumor associated antigen. In one embodiment, the first, second, third and / or fourth Fab specifically bind to a tumor associated antigen.

[0384] In one embodiment, the first and / or second antigen is HER2. In one embodiment, the first and / or second antigen is EGFR. In one embodiment, the first antigen is HER2 and the second antigen is EGFR. In one embodiment, the first antigen is EGFR and the second antigen is HER2.

[0385] In one embodiment, the first and / or second targeting moiety is capable of competing for binding to an epitope on human HER2 with an antibody comprising a VH of SEQ ID NO: 9 and a VL of SEQ ID NO: 10. In one embodiment, the first and / or second targeting moiety comprises a VH domain that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9 and a VL domain that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10.

[0386] In one embodiment, the first and / or second targeting moiety is capable of competing for binding to an epitope on human EGFR with an antibody consisting of a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 12. In one embodiment, the first and / or second targeting moiety comprises a VH domain that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11 and a VL domain that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12.

[0387] In one embodiment, the second antigen that binds to the second binding site of the set of antigen-binding molecules of the present disclosure is an antigen expressed on immune cells such as T cells, more specifically, cytotoxic T cells. In one embodiment, the second binding site binds to an antigen expressed on immune cells such as T cells, more specifically, cytotoxic T cells. In one embodiment, the antigen is CD3.

[0388] In one embodiment, the second antigen is CD3. In one embodiment, the second antigen is human CD3. In one embodiment, the second antigen is CD3ε. In one embodiment, the second antigen is human CD3ε. In one embodiment, CD3 is monovalently bound by a set of antigen binding molecules according to the present disclosure. In one embodiment, neither the first nor the second antigen binding molecule according to the present disclosure binds to CD3 alone. In one embodiment, neither the VH nor the VL of the second binding site according to the present disclosure binds to CD3 alone.

[0389] In one embodiment, the second binding site is specific for CD3, particularly human CD3. In one embodiment, the antibody Fv domain is specific for CD3, particularly human CD3. In one embodiment, the second antigen is human CD3ε comprising SEQ ID NO: 57. In one embodiment, the second antigen is human CD3ε comprising SEQ ID NO: 58. In one embodiment, the second antigen is the extracellular region of human CD3ε comprising SEQ ID NO: 57.

[0390] In one embodiment, the extracellular region of human CD3ε has the amino acid sequence: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO:57). Human CD3ε, including the signal sequence, has the amino acid sequence according to UniProt P07766: [ka] has.

[0391] In one embodiment, the second binding site of the first and second antigen-binding molecules specifically binds to CD3, particularly human CD3, more particularly human CD3ε. In one embodiment, the second binding site formed by the first and second antigen-binding molecules in the set of antigen-binding molecules of the present disclosure specifically binds to CD3, particularly human CD3, more particularly human CD3ε. In one embodiment, the second binding site is an antibody Fv domain. In one embodiment, the antibody Fv domain is specific for CD3. In one embodiment, the antibody Fv domain is specific for CD3ε. In one embodiment, the antibody Fv domain is specific for human CD3ε.

[0392] In one embodiment, the Fv domain is formed by non-covalent association of either the VH or VL domain of a first antigen-binding molecule with a complementary VH or VL domain of a second antigen-binding molecule in a set of antigen-binding molecules of the present disclosure.

[0393] In one embodiment, CD3 is monovalently bound by the set of antigen binding molecules according to the present disclosure. In one embodiment, the set of antigen binding molecules according to the present disclosure is monovalently bound to CD3. In one embodiment, the second binding site competes with a monoclonal antibody specific for CD3 for binding to an epitope of CD3, in particular CD3ε. In one embodiment, the second binding site may compete with any one of the antibodies specific for CD3 disclosed in the present application. In one embodiment, the second binding site may compete with any one of the antibodies specific for CD3 disclosed in WO2022 / 063819, which is incorporated herein in its entirety. In an embodiment of the present disclosure, the second binding site comprises any of the VH and / or VL domains disclosed in WO2022 / 063819.

[0394] In one embodiment, the second binding site can compete with any one of the antibodies specific for CD3 disclosed in Table 2 or Table 3 of the present disclosure for binding to an epitope of CD3.

[0395] In one embodiment, the second binding site present in the set of antigen binding molecules according to the present disclosure may compete for binding to an epitope of CD3 with an antibody comprising a VH domain of SEQ ID NO: 1 and a VL domain of SEQ ID NO: 2. In one embodiment, the second binding site present in the set of antigen binding molecules according to the present disclosure may compete for binding to an epitope of CD3 with an antibody comprising a VH domain of SEQ ID NO: 59 and a VL domain of SEQ ID NO: 60.

[0396] In one embodiment, the VH domain of the second binding site specific for CD3 comprises a VH of SEQ ID NO: 1 and a VL of SEQ ID NO: 2. In one embodiment, the VH domain of the second binding site specific for CD3 comprises a VH of SEQ ID NO: 59 and a VL of SEQ ID NO: 60.

[0397] In one embodiment, the second binding site specific for CD3 comprises a VH and VL domain comprising: a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO:3; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO:4; c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO:5; d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO:6; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7; and f) An LCDR3 region comprising the amino acid sequence of SEQ ID NO:8.

[0398] In one embodiment, the second binding site specific for CD3 comprises a VH and VL domain comprising: a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 61; b) an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 62; c) an HCDR3 region comprising the amino acid sequence of SEQ ID NO:5; d) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 63; e) an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7; and f) An LCDR3 region comprising the amino acid sequence of SEQ ID NO:8.

[0399] In one embodiment, a first antigen binding molecule according to the present disclosure comprises a VH domain of a second binding site specific for CD3 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 59. In one embodiment, a second antigen binding molecule according to the present disclosure comprises a VH domain of a second binding site specific for CD3 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 59.

[0400] In one embodiment, the first antigen binding molecule according to the present disclosure comprises a VH domain of a second binding site specific for CD3 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 60. In one embodiment, the second antigen binding molecule according to the present disclosure comprises a VL domain of a second binding site specific for CD3 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 60.

[0401] In one embodiment, the first antigen binding molecule according to the present disclosure comprises a VL domain of a second binding site specific for CD3 comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:59, and the second antigen binding molecule according to the present disclosure comprises a VL domain of a second binding site specific for CD3 comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:60.

[0402] In one embodiment, the first antigen binding molecule according to the present disclosure comprises a VL domain of a second binding site specific for CD3 comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:60, and the second antigen binding molecule according to the present disclosure comprises a VH domain of a second binding site specific for CD3 comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:59.

[0403] In one embodiment, the first antigen binding molecule according to the present disclosure comprises a VH domain of a second binding site specific for CD3 comprising: (a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 4, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 5; or (b) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 61, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 62, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 5.

[0404] In one embodiment, the first antigen binding molecule according to the present disclosure comprises a VL domain of a second binding site specific for CD3, comprising: (a) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 6; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 8; or (b) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 63, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 8.

[0405] In one embodiment, the second antigen binding molecule according to the present disclosure comprises a VH domain of a second binding site specific for CD3 comprising: (a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 3, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 4, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 5; or (b) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 61, an HCDR2 region comprising the amino acid sequence of SEQ ID NO: 62, and an HCDR3 region comprising the amino acid sequence of SEQ ID NO: 5.

[0406] In one embodiment, the second antigen binding molecule according to the present disclosure comprises a VL domain of a second binding site specific for CD3, comprising: (a) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 6; an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 8; or (b) an LCDR1 region comprising the amino acid sequence of SEQ ID NO: 63, an LCDR2 region comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 region comprising the amino acid sequence of SEQ ID NO: 8.

[0407] In one embodiment, the Fv region specific for CD3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:59 and a VL domain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:60.

[0408] In one embodiment, the set of antigen-binding molecules according to the present disclosure can simultaneously bind two target cell antigens, in particular a tumor-associated antigen expressed on the same cancer cell and CD3 expressed on a T cell. In such an embodiment, the target cell binds bivalently and the T cell binds monovalently.

[0409] In one embodiment, the set of antigen binding molecules according to the present disclosure can crosslink T cells and target cells by simultaneously binding to two target cell antigens and CD3. In one embodiment, the set of antigen binding molecules according to the present disclosure can crosslink T cells and target cells by simultaneously binding to two different target cell antigens and CD3. In one embodiment, such simultaneous binding results in lysis of the target cells, particularly lysis of tumor cells. In one embodiment, such simultaneous binding results in activation of the T cells. In one embodiment, simultaneous binding results in a cellular response of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers.

[0410] In one embodiment, the set of antigen binding molecules according to the present disclosure can redirect the cytotoxic activity of T cells to target cells when the set of antigen binding molecules bind to their target antigens on the target cells. The T cells according to any of the embodiments according to the present disclosure are cytotoxic T cells. In one embodiment, the T cells are CD4+ or CD8+ T cells.

[0411] The on-cell or on-target antigen forming trivalent or tetravalent trispecific antibody realized by the set of antigen binding molecules according to the present disclosure allows monovalent or bivalent binding to the first and third antigens expressed on target cells, such as cancer cells, and monovalent binding to the second antigen, such as CD3 on T cells. The newly formed antibodies combine high affinity binding and avidity effects to the first and third antigens, resulting in significant differences in binding affinity to CD3 and the target antigen. The set of antigen binding molecules according to the present disclosure is particularly suitable for targeting different target antigens. However, in some cases, it may be beneficial to target only one target antigen and thus have specificity for the same antigen.

[0412] Fc area The Fc region of the antigen-binding molecule of the present disclosure is composed of a pair of polypeptides comprising a conventional immunoglobulin heavy chain domain. The Fc region of a conventional IgG exists as a dimer, each subunit of which comprises a CH2 and a CH3 IgG heavy chain constant domain. The two Fc region subunits can stably bind to each other. Thus, in one embodiment, the two Fc region subunits of the antigen-binding molecule of the present disclosure are capable of stably binding to each other. In one embodiment, the Fc region of the antigen-binding molecule of the present disclosure is an IgG Fc region. In one embodiment, the Fc region is an IgG1 Fc region. In one embodiment, the Fc region is of human origin. In one embodiment, the Fc region is a human IgG1 Fc region.

[0413] The two Fc region subunits of the antigen-binding molecule according to the present disclosure are typically composed of two non-identical polypeptide chains. To improve the yield and purity of the molecule in recombinant production, it is advantageous to introduce one or more modifications in the Fc region that promote binding of the two non-identical polypeptides that form the Fc region subunits. Thus, in certain embodiments, the present disclosure provides a heterodimeric antigen-binding molecule that relies on the use of two different mutant Fc region subunits that self-assemble to form a heterodimeric molecule. In one embodiment, the Fc region of the antigen-binding molecule according to the present disclosure comprises one or more modifications that promote binding of the first and second Fc region subunits. In one embodiment, the first and second Fc region subunits and / or the third and fourth Fc region subunits of the antigen-binding molecule according to the present disclosure comprise one or more modifications that promote binding of the first and second Fc region subunits and / or the third and fourth Fc region subunits.

[0414] In one embodiment, the first Fc region subunit and the second Fc region subunit comprise one or more modifications that reduce homodimerization or reduce homodimerization between two identical polypeptide chains comprising the same Fc region subunit. In one embodiment, the third Fc region subunit and the fourth Fc region subunit comprise one or more modifications that reduce homodimerization or reduce homodimerization between two identical polypeptide chains comprising the same Fc region subunit.

[0415] In one embodiment, the first and second Fc region subunits comprise different amino acid modifications such that the first Fc region of the heterodimer is more stable than the Fc region of a homodimer, hi one embodiment, the third and fourth Fc region subunits comprise different amino acid modifications such that the first Fc region of the heterodimer is more stable than the Fc region of a homodimer.

[0416] In one embodiment, the first and second Fc region subunits comprise different amino acid modifications such that binding of the first and second Fc region subunits is enhanced. In one embodiment, the third and fourth Fc region subunits comprise different amino acid modifications such that binding of the third and fourth Fc region subunits is enhanced. In one embodiment, the first or second Fc region is an immunoglobulin Fc region. In one embodiment, the immunoglobulin Fc region is an IgG Fc region. In one embodiment, the IgG Fc region is a human IgG Fc region. In one embodiment, the human IgG Fc region is a human IgG1 region.

[0417] The modification may be present in the first Fc region subunit and / or the second Fc region subunit. The modification may also be present in the third Fc region subunit and / or the fourth Fc region subunit. In one embodiment, such a modification is present in the first and second Fc region subunits. In one embodiment, such a modification is present in the third and fourth Fc region subunits. In one embodiment, such a modification is present in the first and second Fc region subunits and the third and fourth Fc region subunits. In one embodiment, such a modification is present in the CH3 domain of each Fc region subunit. The modification can be achieved by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0418] Typically, in heterodimerization approaches known in the art, the CH3 domain of one polypeptide chain (e.g., an immunoglobulin heavy chain) and the CH3 domain of another polypeptide chain are both engineered to be complementary, such that a polypeptide containing one engineered CH3 domain can no longer homodimerize with another polypeptide chain of the same structure, and is thereby forced to heterodimerize with another polypeptide containing a complementary engineered CH3 domain.

[0419] Several approaches for CH3 modification aimed at promoting heterodimerization are described, for example, in WO 96 / 27011, WO 98 / 050431, EP 1 870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954, WO 2013 / 096291, which are incorporated herein by reference.

[0420] One of these heterodimerization techniques known in the art is the so-called "knobs-into-holes" technique, which is described, for example, in WO 96 / 027011; Ridgway, JB, et al, Protein Eng. 9 (1996) 617-621; Merchant, AM, et al, Nat. Biotechnol. 16 (1998) 677-681; U.S. Pat. Nos. 5,731,168; 7,695,936; WO 98 / 050431; Carter, J Immunol Meth 248, 7-15 (2001), which are incorporated by reference. "Knobs-into-holes" technology broadly involves: (1) mutating the CH3 domain of each Fc region subunit to promote heterodimerization; and (2) combining the mutated Fc region subunits under conditions that promote heterodimerization. "Knobs" or "protrusions" are typically created by substituting small amino acids of the parent antibody with larger amino acids (e.g., T366Y or T366W); "holes" or "voids" are created by substituting larger residues of the parent antibody with smaller amino acids (e.g., Y407T, T366S, L368A and / or Y407V) (using numbering according to the EU index).

[0421] In one embodiment, the modification present in the Fc region of the antigen-binding molecule according to the present disclosure is a "knob-into-hole" modification, comprising a "knob mutation" in one of the two Fc region subunits and a "hole mutation" in the other complementary Fc region subunit. Knob and hole modifications can be achieved by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In one embodiment, the CH3 domain of each Fc region subunit is modified according to the knob-into-hole technique.

[0422] In one embodiment, using numbering according to the EU index, in the CH3 domain of the first and / or third Fc region subunit, the threonine residue at position 366 is substituted with a tryptophan residue (T366W) and in the CH3 domain of the second and / or fourth Fc region subunit, the tyrosine residue at position 407 is substituted with a valine residue (Y407V). In one embodiment, using numbering according to the EU index, in the CH3 domain of the second and / or fourth Fc region subunit, the threonine residue at position 366 is substituted with a serine residue (T366S) and the leucine residue at position 368 is substituted with an alanine residue (L368A).

[0423] In one embodiment, using numbering based on the EU index, the serine residue at position 354 in the CH3 domain of the first and / or third Fc region subunit is replaced with a cysteine ​​residue (S354C), and the tyrosine residue at position 349 in the CH3 domain of the second and / or fourth Fc region subunit is replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues forms a disulfide bridge between the two Fc region subunits, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0424] In a more specific embodiment, the disclosure provides an antigen binding molecule, wherein in the CH3 domain of the first and / or third Fc region subunit, the threonine residue at position 366 is substituted with a tryptophan residue (T366W), the serine residue at position 354 is substituted with a cysteine ​​residue (S354C), and in the CH3 domain of the second and / or fourth Fc region subunit, the tyrosine residue at position 407 is substituted with a valine residue (Y407V), the threonine residue at position 366 is substituted with a serine residue (T366S), the leucine residue at position 368 is substituted with an alanine residue (L368A), and the tyrosine residue at position 349 is substituted with a cysteine ​​residue (Y349C), using numbering according to the EU index.

[0425] In one embodiment of the disclosure, the disclosure provides an antigen binding molecule, wherein either the VH domain or the VL domain of the second binding site and the Fc region subunit comprising the knob mutation are present on the same polypeptide chain.

[0426] In one embodiment of the present disclosure, the present disclosure provides an antigen binding molecule, wherein the complementary VH or VL domain of the second binding site and the Fc region subunit comprising the knob mutation are present on the same polypeptide chain.

[0427] In one embodiment, the Fab heavy chain, either the VH or VL domain of the second binding site, and the Fc region subunit containing the knob mutation are present on the same polypeptide chain.

[0428] In one embodiment, the Fab heavy chain, the complementary VH or VL domain of the second binding site and the Fc region subunit comprising the knob mutation are present on the same polypeptide chain.

[0429] Fc binding The Fc region of an immunoglobulin generally confers favorable pharmacokinetic properties to an antibody, such as an extended half-life in serum, as well as the ability to mediate effector functions through binding to Fc receptors expressed on cells. On the other hand, binding to Fc receptors can also result in undesired activation of certain cell surface receptors that lead to unwanted cytokine release and severe side effects upon systemic administration.

[0430] Thus, in certain embodiments, the Fc region of an antigen binding molecule according to the present disclosure is engineered to have an altered binding affinity for an Fc receptor and / or C1q, or to have an altered effector function, compared to an unengineered or wild-type Fc region.

[0431] The altered effector function may include, but is not limited to, one or more of altered complement dependent cytotoxicity (CDC), altered antibody-dependent cell-mediated cytotoxicity (ADCC), altered antibody-dependent cellular phagocytosis (ADCP). In certain embodiments, the altered effector function is one or more selected from the group consisting of CDC, ADCC, and ADCP. In one embodiment, the altered effector function is ADCC. In one embodiment, the altered effector function is CDC. In one embodiment, the altered effector function is ADCP. In one embodiment, the altered effector function is CDC, ADCC, and ADCP.

[0432] The altered effector function is typically achieved by mutating at least one, preferably both, of the wild-type Fc region subunits. Substitutions that result in increased binding, as well as decreased binding, are also useful. To alter the binding properties of the Fc region, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid with different structure and / or chemical properties, are preferred.

[0433] Fc receptor binding and / or effector function In certain therapeutic situations, it may be desirable to reduce or inhibit normal binding of the Fc region to one or more or all of the Fc receptors and / or binding to complement components such as C1q. For example, it may be desirable to reduce or inhibit binding of the Fc region to one or more or all of the Fcγ receptors (e.g., FcγRl, FcγRIIa, FccγRIIb, FcγRIIIa).

[0434] In particular, when the set of antigen-binding molecules according to the present disclosure simultaneously binds to a receptor of an immune effector cell (such as TCR), it is desirable to block FcγRIIIa binding to eliminate or significantly reduce ADCC activity, and / or block 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 reduced complement-dependent cytotoxicity (CDC), reduced or eliminated antibody-dependent cell-mediated cytotoxicity (ADCC), and reduced or eliminated antibody-dependent cellular phagocytosis (ADCP). In a particular embodiment, 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 function is ADCC. In one embodiment, the reduced or eliminated effector function is CDC. In one embodiment, the reduced or eliminated effector function is ADCP. In an embodiment, the reduced or eliminated effector function is CDC, ADCC, and ADCP.

[0435] In one embodiment, the Fc region of the antigen binding molecule according to the present disclosure is engineered to have a reduced binding affinity to an Fc receptor and / or C1q and / or a reduced effector function compared to a non-engineered Fc region. In one embodiment...

Claims

1. below: a) from its N-terminus to its C-terminus: i. a first antibody fragment comprising a first binding site specific for a first antigen; ii. a first peptide linker, and iii. Either the VH or VL domain of the Fv region specific for CD3 A first antigen-binding molecule comprising: a first antigen-binding molecule in which the first antibody fragment is fused to the N-terminus of either the VH domain or the VL domain of the Fv region via the first peptide linker; b) from its N-terminus to its C-terminus: i. a second antibody fragment comprising a third binding site specific for a third antigen; ii. a second peptide linker; iii. A first Fc region composed of a first Fc region subunit and a second Fc region subunit, each Fc region subunit composed of a CH2 domain and a CH3 domain; iv. a third peptide linker, and v. A complementary VH domain or a complementary VL domain of the Fv region A second antigen-binding molecule comprising: the second antibody fragment is fused to the N-terminus of the first Fc region subunit via the second peptide linker; the N-terminus of the complementary VH domain or complementary VL domain of the Fv region is fused to the C-terminus of the first Fc region subunit via the third peptide linker; a second antigen-binding molecule, wherein the N-terminus of the second Fc region subunit is fused to a fourth peptide linker; A set of antigen-binding molecules comprising: the first antigen-binding molecule and the second antigen-binding molecule are not linked by a covalent bond; A set of antigen-binding molecules.

2. The first antigen-binding molecule further comprises: i. a fifth peptide linker; ii. A second Fc region composed of a third Fc region subunit and a fourth Fc region subunit, each Fc region subunit composed of a CH2 domain and a CH3 domain; Including, the C-terminus of either the VH domain or the VL domain of the Fv region is fused to the N-terminus of the third Fc region subunit via the fifth peptide linker; the N-terminus of the fourth Fc region subunit is fused to a sixth peptide linker; A set of antigen-binding molecules according to claim 1.

3. The second antigen-binding molecule further comprises: a) a third antibody fragment comprising a fourth binding site specific for said third antigen; Including, the third antibody fragment is fused to the N-terminus of the second Fc region subunit via the fourth peptide linker; A set of antigen-binding molecules according to claim 1.

4. the first antibody fragment is a first Fab, the second antibody fragment is a second Fab, and the third antibody fragment is a third Fab; A set of antigen-binding molecules according to claim 1.

5. the C-terminus of the first Fab heavy chain is fused to the N-terminus of either the VH domain or the VL domain of the Fv region via the first peptide linker; A set of antigen-binding molecules according to claim 4.

6. the C-terminus of the second Fab heavy chain is fused to the N-terminus of the first Fc region subunit via the second peptide linker; A set of antigen-binding molecules according to claim 4.

7. the C-terminus of the third Fab heavy chain is fused to the N-terminus of the second Fc region subunit via the fourth peptide linker; A set of antigen-binding molecules according to claim 4.

8. The first antigen-binding molecule comprises a first polypeptide and a second polypeptide, a) the first polypeptide comprises the light chain of the first Fab; b) said second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of the first Fab; ii. the first peptide linker; iii. either the VH domain or the VL domain of the Fv region specific for CD3 Including, A set of antigen-binding molecules according to claim 4.

9. The first antigen-binding molecule comprises a first polypeptide, a second polypeptide, and a third polypeptide, a) the first polypeptide comprises the light chain of the first Fab; b) said second polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of the first Fab; ii. the first peptide linker; iii. either the VH domain or the VL domain of the Fv region specific for CD3; iv. the fifth peptide linker, and v. the third Fc region subunit, which is composed of, from its N-terminus to its C-terminus, a CH2 domain and a CH3 domain; Including, c) the third polypeptide comprises from its N-terminus to its C-terminus: i. the sixth peptide linker; ii. the fourth Fc region subunit, which is composed of, from its N-terminus to its C-terminus, a CH2 domain and a CH3 domain; Including, A set of antigen-binding molecules according to claim 4.

10. The second antigen-binding molecule comprises a fourth polypeptide, a fifth polypeptide, and a sixth polypeptide, a) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. the fourth peptide linker; ii. the second Fc region subunit, which is composed of a CH2 domain and a CH3 domain from its N-terminus to its C-terminus; Including, b) the fifth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of the second Fab; ii. the second peptide linker; iii. The first Fc region subunit, which is composed of a CH2 domain and a CH3 domain from its N-terminus to its C-terminus. iv. the third peptide linker; v. A complementary VH domain or a complementary VL domain of the Fv region specific for CD3 Including, c) the sixth polypeptide comprises the light chain of the second Fab; A set of antigen-binding molecules according to claim 4.

11. The second antigen-binding molecule comprises a fourth polypeptide, a fifth polypeptide, a sixth polypeptide, and a seventh polypeptide, a) the fourth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of the third Fab; ii. the fourth peptide linker; iii. The second Fc region subunit, which is composed of a CH2 domain and a CH3 domain from its N-terminus to its C-terminus. Including, b) the fifth polypeptide comprises from its N-terminus to its C-terminus: i. the heavy chain of the second Fab ii. the second peptide linker iii. the first Fc region subunit, which is composed of a CH2 domain and a CH3 domain from its N-terminus to C-terminus; iv. the third peptide linker v. A complementary VH domain or a complementary VL domain of the Fv region Including, c) the sixth polypeptide comprises the light chain of the second Fab; d) the seventh polypeptide comprises the light chain of the third Fab; A set of antigen-binding molecules according to claim 4.

12. Neither the first antigen-binding molecule nor the second antigen-binding molecule can bind to CD3 alone. A set of antigen-binding molecules according to claim 1.

13. Either the VH domain or the VL domain of the Fv region of the first antigen-binding molecule and the complementary VH domain or the complementary VL domain of the Fv region of the second antigen-binding molecule can bind to each other non-covalently, thereby forming the Fv region specific for CD3. A set of antigen-binding molecules according to claim 1.

14. The set of antigen-binding molecules according to claim 1, wherein the peptide linker has a length of 5 to 49 amino acid residues, preferably 5 to 29 amino acid residues.

15. The set of antigen-binding molecules according to claim 1 , wherein the first antigen and the third antigen are present on the same cell, and the CD3 is present on a different cell.

16. The set of antigen-binding molecules according to claim 1 , wherein the first antigen and the third antigen are different.

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