Novel antigen binding molecular forms

Abstract

A novel antigen binding molecular form is provided. Comprising a non-naturally configured Fab domain, ABM conjugates comprising the ABM and a cytotoxic or cytostatic agent, pharmaceutical compositions containing the ABM and ABM conjugates, methods of using the ABM, ABM conjugates and pharmaceutical compositions for the treatment of cancer, nucleic acids encoding the ABM, and methods of using the ABM conjugates. A cell engineered to express the ABM and a method of producing the ABM.

Description

[0001] This application is a divisional application of Chinese patent application CN202080069722.6, filed on August 7, 2020, entitled "Novel Antigen-Binding Molecular Form".

[0002] Cross-reference to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 884,496, filed August 8, 2019, and U.S. Provisional Application No. 63 / 050,483, filed July 10, 2020, the contents of each of which are incorporated herein by reference in their entirety.

[0004] sequence list

[0005] This application contains a sequence list, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on August 6, 2020, is named RGN-001WO_SL.txt and is 25,358 bytes in size. Technical Field

[0006] This invention specifically relates to a novel antigen-binding molecule. Background Technology

[0007] Most naturally occurring antibody molecules typically consist of two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each of the heavy and light chain polypeptides contains a variable domain (variable region) (typically the amino-terminal portion of the polypeptide chain), which includes a binding region capable of interacting with the antigen. Each of the heavy and light chain polypeptides also includes a constant region (typically the carboxyl-terminal portion).

[0008] Recombinant monoclonal antibodies, derived from a single clone of a cell or cell line, have become a highly successful class of biological drugs for treating a wide variety of diseases over the past two decades. Monoclonal antibodies (mAbs) are an important class of biological therapeutics and have achieved remarkable success in treating many life-threatening chronic diseases.

[0009] An increase in affinity and / or cohesion occurs when the antigen-binding portion of an antibody (e.g., the Fab domain) is close to an epitope. However, the geometry of conventional antibody forms limits the ability of antibodies to recognize multiple epitopes on a single target molecule, especially when the target size is small, or when the desired epitopes (containing epitopes on multiple target molecules) are physically relatively close, or are expected to become physically close. Therefore, it would be useful to develop efficient platforms for generating binding molecules that may improve affinity, cohesion, or antibody function through alternative antibody-antigen binding geometries. Summary of the Invention

[0010] This disclosure provides antigen-binding molecules (“ABMs”) containing at least two Fab domains of at least two non-natural configurations. The ABM comprises at least two polypeptide chains, each polypeptide chain including an Fc domain and a component of the at least two Fab domains. Exemplary ABMs of this disclosure are described in... Figure 1B , 2B And shown in 3A to 3D.

[0011] Each polypeptide chain, including the Fc domain and any associated polypeptide chain, is referred to herein as a “half-antibody.” A typical ABM of this disclosure comprises two half-antibodies associated through their Fc domains. The associated Fc domains together form an Fc region. In addition to the Fc region, each half-antibody of the typical ABM of this disclosure includes at least one Fab domain of a non-native conformation. At least one or both of the Fab domains of the non-native conformation bind to the target molecule. “Natural conformation” or “native immunoglobulin conformation” refers to the conformation of antibody domains in naturally occurring IgG antibodies. In the accompanying schematic diagram, the VH domain is labeled with the number (1), the CH1 domain with the number (2), the hinge domain with the number (3), the CH2 domain with the number (4), the CH3 domain with the number (5), the VL domain with the number (6), the CL domain with the number (7), and the linker of the non-hinge domain with the number (8). Therefore, referring to the markings in the attached diagram, the main conformations of natural immunoglobulins are as follows:

[0012] ●The first (heavy chain) polypeptide is basically composed of the following in the N to C orientation: VH domain (1), CH1 domain (2), hinge region (3), which is connected to the hinge region of the second (heavy chain) polypeptide by disulfide bond; CH2 domain (4) and CH3 domain (5).

[0013] ●The second (heavy chain) polypeptide is basically composed of the following in the N to C orientation: VH domain (1), CH1 domain (2), hinge region (3), which is connected to the hinge region of the first (heavy chain) polypeptide by disulfide bonds; CH2 domain (4) and CH3 domain (5).

[0014] ●The third (light chain) polypeptide, which is essentially composed of the following in its N-to-C-terminal orientation: a VL domain (6) and a CL domain (7), both of which associate with the first (heavy chain) polypeptide; and

[0015] ●The fourth (light chain) polypeptide, which is essentially composed of the following in its N-to-C-terminal orientation: VL domain (6) and CL domain (7), which associate with the second (heavy chain) polypeptide.

[0016] The use of terms like "native configuration" or "native immunoglobulin configuration" is not intended to limit the term to wild-type antibody sequences or monospecific antibodies only. Rather, as... Figure 1A and Figure 2A As shown, this form can be applied to monospecific antibodies ( Figure 1A ) or traditional bispecific antibodies with variant sequences ( Figure 2B ). Figure 1A monospecific antibody forms and Figure 2A The fundamental difference between bispecific antibody forms is not their conformation, but the use of Fc heterodimers (e.g., as described in Section 6.2.7.2), where each Fc region is linked to a different VH domain, thereby allowing binding to different epitopes. For clarity, as used herein, the term “bispecific” refers to binding to any two different epitopes, whether on the same antigen or target molecule or on different antigens or target molecules.

[0017] The ABM disclosed herein is particularly useful for binding small, soluble target molecules, such as cytokines or chemokines, and finds applications in antagonizing the activity of target molecules, for example by blocking the binding of target molecules to binding couplers (such as receptors). Not bound by theory, it is believed that the binding forms of this disclosure allow for binding of target molecules with greater affinity and / or affinity than that of native immunoglobulins comprising at least two identical Fab domains.

[0018]

[0019] These ABM forms are described in more detail below.

[0020] In a first aspect, the ABM disclosed herein includes:

[0021] ●The first half-antibody, wherein the first half-antibody includes the following orientation from the N to the C terminus:

[0022] -Optional hinge structure domain;

[0023] - The first Fc structural domain; and

[0024] - A first Fab (Fab1) structural domain, the first Fab structural domain including a first heavy chain variable region (VH) associated with a first light chain variable region (VL); and

[0025] ●The second half-antibody, wherein the N-to-C-terminal orientation of the second half-antibody includes:

[0026] -Optional hinge structure domain;

[0027] - The second Fc structural domain; and

[0028] - A second Fab (“Fab2”) domain, the second Fab domain including a second VH associated with a second VL.

[0029] The first Fc structural domain and the second Fc structural domain associate with each other to form an Fc region, and the optional hinge structural domain, if present, can associate with each other via disulfide bonds.

[0030] Figure 1B and Figure 2B Two embodiments of this type of ABM are shown herein, which are generally referred to herein as ABM form "A" ("Type A") and sometimes as "Fc-Fab" form, and are depicted in Figure 13A , Figure 13B and Figure 13C Variations thereof. Therefore, this disclosure provides depictions of... Figure 1B and Figure 2B Type A ABM includes:

[0031] ● First polypeptide, the first polypeptide comprising, in the N-to-C-terminal orientation:

[0032] - Optional hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond;

[0033] -Fc domain, the Fc domain includes CH2 domain (4) and CH3 domain (5).

[0034] - Optional hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond;

[0035] - Connector (8); and

[0036] -The heavy chain component of the Fab1 domain, comprising the Fab1 VH domain (1) and the Fab1 CH1 domain (2) associated with the light chain component of the Fab1 domain, wherein the light chain component in polypeptide form comprises the Fab1 VL domain (6) and the Fab1 CL domain (7) oriented from N to C; and

[0037] ●The second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0038] - Optional hinge domain (3), which is connected to the hinge domain in the first polypeptide via a disulfide bond;

[0039] - Second Fc domain, which includes CH2 domain (4) and CH3 domain (5).

[0040] - Optional hinge domain (3), which is connected to the hinge domain in the first polypeptide via a disulfide bond;

[0041] - Connector (8); and

[0042] -The heavy chain component of the Fab2 domain includes the Fab2 VH domain (1) and the Fab2 CH1 domain (2) associated with the light chain component of the Fab2 domain, and the light chain component in polypeptide form includes the Fab2 VL domain (6) and the Fab2 CL domain (7) in the N-to-C-terminal orientation.

[0043] The first Fc structural domain and the second Fc structural domain associate with each other to form the Fc region.

[0044] exist Figure 1B In the embodiments described, the two half-antibodies are identical, containing Fc domains that form Fc homodimers, and the resulting ABM is single-specific. Figure 2B In some embodiments, ABM includes Fc heterodimers, thereby allowing the use of different Fab1 and Fab2 VH domains and producing multispecific, such as bispecific, molecules. Although Figure 1B , Figure 2B and 13A An embodiment of the ABM is shown, in which a hinge region is formed by a hinge structure domain at the N end of the Fc structure domain. However, type A ABMs may not have a hinge region (not shown) or may have a hinge region at the C end of the Fc region. Figure 13C ) or a hinge area with N and C ends of the Fc region ( Figure 13B ).like Figures 13A-13C The exemplary hinge domains depicted that can be used at the N-terminus and / or C-terminus of the Fc region include the amino acid sequences GGGGSCPPC (SEQ ID NO: 1) and ESKYGPPCPPC (SEQ ID NO: 2), but type A ABM can also have alternative hinge region sequences. Similarly, although... Figures 13A-13C It depicts (G4S). n The connector (G4S is disclosed as SEQ ID NO: 3) can be used, but other connector sequences may be used.

[0045] Although Figure 1B and Figure 2B An embodiment of a type A ABM containing only two bonding domains (Fab1 and Fab2) is shown, but the ABM of this disclosure may contain additional bonding domains, such as scFv or Fab domains. However, in some respects, Fab1 and Fab2 are the only bonding domains of the type A ABM.

[0046] In a second aspect, the ABM disclosed herein includes:

[0047] ●The first half-antibody, wherein the first half-antibody includes the following orientation from the N to the C terminus:

[0048] - A first Fab (Fab1) structural domain, the first Fab structural domain including a first VH associated with a first VL;

[0049] - First spacer structural domain; and

[0050] - The first Fc structural domain; and

[0051] ●The second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0052] - A second Fab (Fab2) structural domain, the second Fab structural domain including a second VH associated with a second VL;

[0053] - Second spacer structure domain; and

[0054] -Second Fc structural domain; and

[0055] The first Fc structural domain and the second Fc structural domain associate with each other to form the Fc region.

[0056] Unbound by theory, it is believed that including a spacer domain between the Fc and Fab domains results in greater flexibility between the antigen-binding sites of the Fc and Fab regions, and thus produces higher affinity and / or cohesion of ABMs to their antigens or target molecules. The terms “antigen” and “target molecule” are used interchangeably herein.

[0057] In some embodiments, the spacer structure domain is an extension joint. Figure 3A This ABM form is illustrated herein and is generally referred to herein as form "B" ("Type B") and sometimes as the "extended form". Therefore, this disclosure provides a depiction of... Figure 3A An embodiment of type B ABM includes:

[0058] ● First polypeptide, the first polypeptide comprising, in the N-to-C-terminal orientation:

[0059] -The heavy chain component of the Fab1 domain includes the Fab1 VH domain (1) and the Fab1 CH1 domain (2) associated with the light chain component of the Fab1 domain, and the light chain component in polypeptide form includes the Fab1 VL domain (6) and the Fab1 CL domain (7) in the N-to-C-terminal orientation.

[0060] - Connector structural domain (8), the connector structural domain being an extension connector;

[0061] - Hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond; and

[0062] - A first Fc domain, comprising a CH2 domain (4) and a CH3 domain (5); and

[0063] ●The second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0064] -The heavy chain component of the Fab2 domain includes the Fab2 VH domain (1) and the Fab2 CH1 domain (2) associated with the light chain component of the Fab2 domain, and the light chain component in polypeptide form includes the Fab2 VL domain (6) and the Fab2 CL domain (7) in the N-to-C-terminal orientation.

[0065] - Connector structural domain (8), the connector structural domain being an extension connector;

[0066] - Hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond; and

[0067] - Second Fc domain, which includes CH2 domain (4) and CH3 domain (5).

[0068] Although Figure 3A The embodiment of the B-type ABM depicted contains only two bonding domains (Fab1 and Fab2), but the B-type ABM of this disclosure may contain additional bonding domains, such as scFv or Fab domains. However, in some respects, Fab1 and Fab2 are the only bonding domains of the B-type ABM of this disclosure.

[0069] In other embodiments, the spacer domain is a Fab domain. Figure 3B-3D Different variants of this ABM form are shown in the document, referred to herein as form "C" ("C-type"). Therefore, the C-type ABM comprises a third Fab (Fab3) structural domain and a fourth Fab (Fab4) structural domain, with the following configuration:

[0070] ●The first hemibody, wherein the first hemibody includes, in the N-to-C-terminal orientation...

[0071] - A first Fab (Fab1) structural domain, the first Fab structural domain including a first VH associated with a first VL;

[0072] - A third Fab (Fab3) structural domain, the third Fab structural domain including a third VH associated with a third VL; and

[0073] - The first Fc structural domain; and

[0074] ●The second half-antibody, wherein the N-to-C-terminal orientation of the second half-antibody includes:

[0075] - A second Fab (Fab2) structural domain, the second Fab structural domain including a second VH associated with a second VL;

[0076] - A fourth Fab (Fab4) structural domain, the fourth Fab structural domain including a fourth VH associated with a fourth VL; and

[0077] -Second Fc structural domain.

[0078] Therefore, this disclosure provides a description of... Figure 3B-3D An embodiment of type C ABM includes:

[0079] ● First polypeptide, the first polypeptide comprising, in the N-to-C-terminal orientation:

[0080] -The heavy chain component of the Fab1 domain includes the Fab1 VH domain (1) and the Fab1 CH1 domain (2) associated with the light chain component of the Fab1 domain, and the light chain component in polypeptide form includes the Fab1 VL domain (6) and the Fab1 CL domain (7) in the N-to-C-terminal orientation.

[0081] - Connector structural domain (8);

[0082] -The heavy chain component of the Fab3 domain includes the Fab3 VH domain (1) and the Fab3 CH1 domain (2) associated with the light chain component of the Fab3 domain, and the light chain component in polypeptide form includes the Fab3 VL domain (6) and the Fab3 CL domain (7) in the N-to-C-terminal orientation.

[0083] - Hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond; and

[0084] - A first Fc domain, comprising a CH2 domain (4) and a CH3 domain (5); and

[0085] ●The second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0086] -The heavy chain component of the Fab2 domain includes the Fab2 VH domain (1) and the Fab2 CH1 domain (2) associated with the light chain component of the Fab2 domain, and the light chain component in polypeptide form includes the Fab2 VL domain (6) and the Fab2 CL domain (7) in the N-to-C-terminal orientation.

[0087] - Connector structural domain (8);

[0088] -The heavy chain component of the Fab4 domain includes the Fab4 VH domain (1) and the Fab4 CH1 domain (2) associated with the light chain component of the Fab4 domain, and the light chain component in polypeptide form includes the Fab4 VL domain (6) and the Fab4 CL domain (7) in the N-to-C-terminal orientation.

[0089] - Hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond; and

[0090] - Second Fc domain, the second Fc domain includes CH2 domain (4) and CH3 domain (5); wherein the first Fc domain and the second Fc domain associate with each other to form an Fc region.

[0091] Although Figure 3B-3D The embodiments of the C-type ABM depicted contain four bonding domains (Fab1, Fab2, Fab3, and Fab4), but the C-type ABM of this disclosure may contain additional bonding domains, such as scFv or Fab domains. However, in some respects, Fab1, Fab2, Fab3, and Fab4 are the only bonding domains of the C-type ABM of this disclosure.

[0092] The Fab3 and Fab4 domains of a C-type ABM can be non-bonded (e.g., Figure 3B (as shown) or combined (such as) Figure 3C and Figure 3D (As shown). Those embodiments where Fab3 and Fab4 are non-jointed are generally referred to herein as type C1 ABMs, and this form is sometimes referred to herein as a "clamp" form. Those embodiments where Fab3 and Fab4 are joined are generally referred to herein as type C2 ABMs, and this form is sometimes referred to herein as a "tandem Fab" form. The term "2+2 tandem Fab" refers to... Figure 3C and Figure 3D In the illustrated embodiment, Fab1, Fab2, Fab3, and Fab4 are the only binding structural domains in the tandem Fabs. Each of the C1-type and C2-type ABMs can be either a homodimer or a heterodimer.

[0093] In some embodiments of C1-type ABM, the Fab1 and Fab2 domains are different (e.g., binding different epitopes, whether on the same target molecule or different target molecules), and the Fab3 and Fab4 domains are the same non-binding domains. In other embodiments, the Fab3 and Fab4 domains are different non-binding domains.

[0094] In some embodiments of the C2 type ABM, the Fab1 and Fab3 structural domains include the same VH structural domain, and the Fab2 and Fab4 structural domains include the same VH structural domain, such as... Figure 3C As shown. This configuration is referred to as configuration 1, or 1-1-2-2 configuration. In an alternative embodiment of the C2 type ABM, the Fab1 and Fab2 domains include the same VH domain, and the Fab3 and Fab4 domains include the same VH domain, as shown. Figure 3D As shown. This configuration is called configuration 2, or 1-2-1-2 configuration.

[0095] A complete ABM is formed by the association of two half-antibodies through two Fc domains to form an Fc region. When the two half-antibodies are not identical, for example when Fab1 and Fab2 contain different VH domains, Fc heterodimerization methods (e.g., as described in Section 6.2.7.2) can be used to facilitate correct half-antibody pairing and / or their purification. Examples of heterodimerization methods are star mutations (as described in Section 6.2.7.2) or intrawell toggle mutations.

[0096] Although Figure 2B , 3A Figures 3B and 3C illustrate ABMs comprising distinct VH domains in each half-antibody paired via Fc heterodimers, but this form can also be used for Fc homodimers. For example, although Figure 2B and Figure 3A Examples of type A and type B ABMs are shown, each incorporating an Fc heterodimer, which allows for the incorporation of different VH domains in Fab1 and Fab2 to generate multispecific (e.g., bispecific) binding molecules. However, this form can also be used for single-specific type A and type B ABMs having an Fc homodimer and the same VH domains. Similarly, an Fc homodimer can be used to generate single-specific type C ABMs having the same Fab1, Fab2, Fab3, and Fab4 VH domains, or the same Fab1 and Fab2 VH domains and non-binding Fab3 and Fab4 VH domains.

[0097] Furthermore, when the first and second peptides contain different VH domains, different strategies can be used to allow correct VH-VL pairing in multispecific binding molecules. For example, a common light chain capable of effectively pairing with more than one type of VH domain in an ABM can be used. In such embodiments, the light chain peptides (e.g., light chains associated with Fab1 and Fab2 and, if present, Fab3 and Fab4) can be identical. Alternatively, a single-domain Fab can be used, where the heavy chain components ((1) and (2)) can be expressed fused with the light chain components ((6) and (7)).

[0098] The variations of the ABM of this disclosure shown in Figures 1-3 are not intended to be limiting; except as otherwise provided, the ABM of this disclosure may comprise any combination of the modifications shown in Figures 1-3 and Section 6.2 below. Furthermore, references to the first or second polypeptide chain or the left or right hemiantibody are for convenience only and are not intended to convey that the polypeptide chains or hemiantibodies are generated or assembled in any particular order.

[0099] In some embodiments, the first Fab (Fab1) domain and the second Fab (Fab2) domain of the ABM disclosed herein can each bind the same target molecule, such as a small soluble molecule. The first Fab (Fab1) domain and the second Fab (Fab2) domain can bind the same epitope (e.g., in...). Figure 1B and Figure 3D In the embodiments depicted, or in Figure 3A or Figure 3B In variants where Fab1 and Fab2 both have the same VH domain (not shown) or they can combine different epitopes (e.g., in... Figure 2B , Figure 3A , Figure 3B and Figure 3C In the embodiments depicted, whether on the same target molecule or on different target molecules. In the case where the first Fab (Fab1) domain and the second Fab (Fab2) domain bind different epitopes (e.g., two different epitopes on the same target molecule or on different target molecules), they can be selected so that the Fab can bind to its epitopes simultaneously.

[0100] In some embodiments, such as in a C-type ABM, the ABM of this disclosure may include a third Fab (Fab3) structural domain and a fourth Fab (Fab4) structural domain, such as... Figure 3B , Figure 3C and Figure 3D The third and fourth Fab domains can be non-bonded, as described. Figure 3B What is depicted, or they may be combined, such as Figure 3C and Figure 3D As described. When Fab3 and Fab4 are present, they can each bind to the same or different tabletops bound to the Fab1 and Fab2 domains, respectively. For example, Fab1 and Fab3 can share a tabletop, and Fab2 and Fab4 can share a tabletop, such as... Figure 3C As shown in the embodiments. Alternatively, for example, Fab1 and Fab2 may share a tabletop, and Fab3 and Fab4 may share a tabletop, such as... Figure 3DAs illustrated in the embodiments. As used herein, with respect to C-type ABM, the terms "first and second Fab domains" and "Fab1 and Fab2 domains" generally refer to the Nth-most Fab domains, and references to "third and fourth Fab domains" and "Fab3 and Fab4 domains" generally refer to the inner Fab domains.

[0101] Exemplary antigen-binding molecules of this disclosure, including their components and configurations, and their target molecules, are described in Sections 6.2 and 6.3 below, as well as in “A” Specific Examples 1 to 138 and “B” Specific Examples 1 to 72.

[0102] This disclosure further provides conjugates, such as pharmaceutical conjugates, which include the ABM of this disclosure (for convenience, pharmaceutical conjugates are referred to herein as “antibody-drug conjugates” or “ADCs”). Exemplary features of the conjugates are described below in Section 6.4 and in “A” Specific Example 139 and “B” Specific Example 73.

[0103] This disclosure further provides nucleic acids encoding the ABM of this disclosure. The nucleic acid encoding ABM may be a single nucleic acid (e.g., a vector encoding all polypeptide chains of ABM) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains of ABM). This disclosure further provides host cells and cell lines engineered to express the nucleic acids and ABM of this disclosure. This disclosure further provides methods for generating the ABM of this disclosure. Exemplary nucleic acids, host cells, cell lines, and methods for generating ABM are described below in Section 6.5, Specific Examples 144-145 (“A”), and Specific Examples 75-81 (“B”).

[0104] This disclosure further provides pharmaceutical compositions comprising the ABM and ADC of this disclosure. Exemplary pharmaceutical compositions are described below in Section 6.6, Specific Example 140 (“A”), and Specific Example 74 (“B”).

[0105] This document further provides methods for using the ABMs, conjugates, and pharmaceutical compositions of this disclosure, for example, for treating symptoms associated with the aberrant expression or activity of target molecules to which they are bound. Exemplary methods are described below in Section 6.7, Specific Examples 141-143 (“A”), and Specific Examples 82-85 (“B”). Attached Figure Description

[0106] Figure 1A-1B The exemplary homodimer (monospecific divalent) type A ABM of this disclosure ( Figure 1B ) and the corresponding natural antibody forms ( Figure 1A) Schematic diagram. Legend: (1) = VH; (2) = CH1; (3) = hinge; (4) = CH2; (5) = CH3; (6) = VL; (7) = CL; (8) = joint.

[0107] Figure 2A-2B The heterodimer bispecific type A ABM disclosed herein ( Figure 2B ) and the corresponding traditional bispecific antibody forms ( Figure 2A A schematic diagram of the small antigen (Ag) is shown to illustrate the potential ways in which the bispecific ABM interacts with the small antigen. Legend: (1) = VH; (2) = CH1; (3) = hinge; (4) = CH2; (5) = CH3; (6) = VL; (7) = CL; (8) = linker. An asterisk in one of the CH3 domains indicates that the two CH3 domains are not identical and contain one or more mutations that allow heterodimerization (e.g., in-pore button mutations, star mutations, etc.).

[0108] Figures 3A-3D Exemplary B-type ABM of this disclosure ( Figure 3A ) and the exemplary C-type ABM of this disclosure ( Figure 3B-3D A schematic diagram of the C-type ABM shown. The specific embodiment of the C-type ABM illustrated is an exemplary heterodimer C1-type ABM ( Figure 3B ), and exemplary heterodimer C2 type ABM ( Figure 3C ) and exemplary homodimer C2 type ABM ( Figure 3D In some embodiments, these bispecific ABMs use a common light chain VL-CL. In some embodiments across all forms, VH1-CH1 / VL-CL and VH2-CH1 / VL-CL are Fab fragments derived from mAbs that are non-competitive with the antigen. Figure 3B In the form shown (sometimes referred to in this paper as the "clamp" form), the inner Fab fragment VH3-CH1 / VL-CL does not bind to the target molecule. Figure 3A In the form shown (sometimes referred to in this paper as the “extended” form), the inner Fab is replaced with a flexible long connector. Legend: (1) = VH; (2) = CH1; (3) = hinge; (4) = CH2; (5) = CH3; (6) = VL; (7) = CL; (8) = connector. An asterisk in one of the CH3 domains indicates that the two CH3 are not identical and contain one or more mutations that allow heterodimerization (e.g., in-hole knob mutation, star mutation, etc.).

[0109] Figures 4A-4B : Figure 4A and 4B This is a graph showing the activity of the TSLP parent antibody in the TSLP blocking bioassay. Figure 4AThe dose-response curve of hTSLP was determined using the STAT3-luciferase reporter gene assay in Baf3 cells expressing hIL7R and hTSLPR. Figure 4B The activity of the selected TSLP Ab in the TSLP blocking bioassay was determined. The TSLP Ab was incubated with the Baf3 / hIL7R / hTSLPR / STAT3-luciferase reporter cell line in the presence of 100 pM constant hTSLP. Luciferase activity was measured after 5.5 hours of incubation.

[0110] Figure 5 : Figure 5 This figure demonstrates that the anti-hTSLP bispecific IgG4 Ab exhibits TSLP blocking activity similar to that of the corresponding parental Ab combination. The activities of the anti-hTSLP parental antibody combination and the bispecific IgG4 Ab in the hTSLP blocking bioassay were compared. The TSLP Ab was incubated with the Baf3 / hIL7R / hTSLPR / STAT3-luciferase reporter cell line in the presence of 100 pM hTSLP. Luciferase activity was measured after 5.5 hours of incubation.

[0111] Figures 6A-6C : Figures 6A-6C This is a graph comparing different forms of bispecific anti-hTSLP Abs in TSLP blocking bioassays. Several parental Ab pairs were tested: 30206x30217 ( Figure 6A ), 30206x30230 ( Figure 6B ), 30217x30230 ( Figure 6C In the presence of 100 pM hTSLP, TSLP Ab was incubated together with the Baf3 / hIL7R / hTSLPR / STAT3-luciferase reporter cell line. Luciferase activity was measured after 5.5 hours of incubation. Figures 6A-6C The "2xG4S" and "4xG4S" disclosed in the document are SEQ ID NO: 18 and SEQ ID NO: 19, respectively.

[0112] Figure 7 : Figure 7 This is a fractal diagram showing the individual parent mAb in the presence of hTSLP (REGN4009). Analysis of the anti-TSLP mAb:hTSLP complex (solid line) was performed by asymmetric flow field-flow classification coupled to multi-angle light scattering (A4F-MALS). Fractal diagrams of individual samples from H4H30217P2 (grey dashed line) and hTSLP (black dashed line) are also overlaid. The relative UV absorbance at 215 nm for each sample as a function of retention time is shown, along with the molar mass of the measured resolved peak.

[0113] Figure 8 : Figure 8 This is a fractal diagram showing the parent mAb combination in the presence of hTSLP. Analysis was performed on the anti-TSLP mAb combination:hTSLP complex (solid line) via an asymmetric flow field-flow classification coupled to multi-angle light scattering (A4F-MALS). Fractal diagrams of individual samples from H4H30217P2 (grey dashed line) and hTSLP (black dashed line), as well as the H4H30217P2:hTSLP complex (black dashed line), are also overlaid. The relative UV absorbance at 215 nm for each sample as a function of retention time is shown, along with the molar mass of the measured resolved peak.

[0114] Figure 9 : Figure 9 This is a fractal diagram showing the Fc-Fab bispecific Ab in the presence of hTSLP. Analysis of the anti-TSLP Fc-Fab:hTSLP complex (solid line) was performed by asymmetric flow field-flow classification coupled to multi-angle light scattering (A4F-MALS). Fractal diagrams of individual samples from TS-FC1-eL1 (black dashed line), TS-FC6-eL2 (grey dashed line), and hTSLP (black dashed line) are also overlaid. The relative UV absorbance at 215 nm for each sample as a function of retention time is shown, along with the molar mass of the measured resolved peak.

[0115] Figure 10 : Figure 10 This is a fractal diagram showing the clamp-on bispecific Ab in the presence of hTSLP. Analysis was performed on the anti-TSLP clamp-on:hTSLP complex (solid line) by asymmetric flow field-flow classification coupled to multi-angle light scattering (A4F-MALS). Fractal diagrams of individual samples from TS-CL4-eL1 (black dashed line), TS-CL6-eL1 (grey dashed line), and hTSLP (black dashed line) are also overlaid. The relative UV absorbance at 215 nm for each sample as a function of retention time is shown, along with the molar mass of the measured resolved peak.

[0116] Figure 11 : Figure 11This is a fractal diagram showing the 2+2 tandem Fab:hTSLP bispecific Ab in the presence of hTSLP. Analysis of the anti-TSLP 2+2 tandem Fab:hTSLP complex (solid line) was performed via an asymmetric flow field-flow classification coupled to multi-angle light scattering (A4F-MALS). Fractal diagrams of individual samples from TS-CL2-eL2 (black dashed line), TS-CL3-eL2 (grey dashed line), and hTSLP (black dashed line) are also overlaid. The relative UV absorbance at 215 nm for each sample as a function of retention time is shown, along with the molar mass of the measured resolved peak.

[0117] Figure 12 : Figure 12 This is a graph comparing the activities of anti-hTSLP 30217x30230 bispecific Fc-Fab with different adapter lengths in the TSLP blocking bioassay. TSLP Abs were incubated with the Baf3 / hIL7R / hTSLPR / STAT3-luciferase reporter cell line in the presence of a constant 120 pM hTSLP. Luciferase activity was measured after 5.5 hours of incubation. In the absence of TSLP-blocking Abs, all values ​​were normalized relative to STAT3-luciferase activity and expressed as a percentage of STAT3-Luc activity. Figure 12 The G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5 and (G4S)6 connectors disclosed in the paper are SEQ ID NO: 3, 18, 4, 19, 39 and 38, respectively.

[0118] Figures 13A-13D : Figures 13A-13C These are schematic diagrams of different hinge types. Figure 13A Hinge type 1, which shows hinge sequences ESKYGPPCPPC (SEQ ID NO: 2) and (G4S). n Connector (G4S is disclosed as SEQ ID NO: 3); Figure 13B Hinge type 2, which shows hinge sequences ESKYGPPCPPC (SEQ ID NO: 2) and (G4S). n Connector (G4S is disclosed as SEQ ID NO: 3); Figure 13C Hinge type 3, which shows hinge sequences GGGGSCPPC (SEQ ID NO: 1) and (G4S). n Connector (G4S is disclosed as SEQ ID NO: 3). Figure 13DThis is a graph showing the activity of 30217x30230 Fc-Fab cells with different hinge forms (hinge form 1 with G4S connector (SEQ ID NO: 3), hinge form 2 with G4S connector (SEQ ID NO: 3), hinge form 3 with G4S connector (SEQ ID NO: 3), hinge form 1 with (G4S)4 connector (SEQ ID NO: 19), hinge form 2 with (G4S)4 connector (SEQ ID NO: 19), and hinge form 3 with (G4S)4 connector (SEQ ID NO: 19). TSLP Ab was incubated with the Baf3 / hIL7R / hTSLPR / STAT3-luciferase reporter cell line in the presence of 120 pM hTSLP. Luciferase activity was measured after 5.5 hours of incubation. In the absence of TSLP blocking Abs, all values ​​were normalized relative to STAT3-luciferase activity and expressed as a percentage of STAT3-Luc activity.

[0119] Figure 14 : Figure 14 The pharmacokinetic curves of anti-TSLP bispecific Fc-Fab molecules REGN8759 and REGN8760, hIgG4 isotype control REGN1945, conventional hIgG4 bispecific isotype control H4H21237D, and hFcγ homodimer REGN1627 in WT mice are shown.

[0120] Figure 15 : Figure 15 The molar equivalent pharmacokinetic curves of anti-TSLP Fc-Fab antibodies REGN8759 and REGN8760, hIgG4 isotype control REGN1945, conventional hIgG4 bispecific isotype control H4H21237D, and hFcγ homodimer REGN1627 in WT mice are shown.

[0121] Figures 16A-16C : Figures 16A-16C This graph compares the inhibitory activities of anti-ligand X parental mAbX1, mAbX2, and bispecific Fc-Fab mAbX1 x mAbX2 in ligand X signal transduction bioassays. In the presence of 10 pM ( Figure 16A ), 100pM ( Figure 16B ) or 1nM ( Figure 16C With a constant human ligand X, anti-ligand X Ab was incubated together with an engineered luciferase reporter cell line used for receptor X signaling. Luciferase activity was measured after 5.5 hours of incubation.

[0122] Figures 17A-17C : Figures 17A-17CThis graph compares the inhibitory activities of anti-ligand X parent mAbX2, mAbX3, and bispecific Fc-Fab mAbX2 x mAbX3 in ligand X signal transduction bioassays. In the presence of 10 pM ( Figure 17A ), 100pM ( Figure 17B ) or 1nM ( Figure 17C With a constant human ligand X, anti-ligand X Ab was incubated together with an engineered luciferase reporter cell line used for receptor X signaling. Luciferase activity was measured after 5.5 hours of incubation.

[0123] Figures 18A-18C : Figures 18A-18C This graph compares the inhibitory activities of anti-ligand X parent mAbX1, mAbX3, and bispecific Fc-Fab mAbX1 x mAbX3 in ligand X signal transduction bioassays. In the presence of 10 pM ( Figure 18A ), 100pM ( Figure 18B ) or 1nM ( Figure 18C With a constant human ligand X, anti-ligand X Ab was incubated together with an engineered luciferase reporter cell line used for receptor X signaling. Luciferase activity was measured after 5.5 hours of incubation.

[0124] Figures 19A-19E : Figures 19A-19E This is a fractal diagram showing the complex of ligand X and anti-ligand X antibody. The anti-ligand X antibody combined with ligand X was analyzed by asymmetric flow field-flow fractionation coupled to multi-angle light scattering (A4F-MALS). Fractal diagrams of individual samples from the antibody and ligand X are also overlaid. The relative UV absorbance at 215 nm for each sample as a function of retention time is shown, along with the molar mass of the measured resolved peak. Figure 19A and 19B Fractal diagrams of parental antibodies mAbX1, mAbX2, and ligand X are shown; Figure 19C The fractal diagrams of mAbX1 x mAbX2 Fc-Fab and ligand X are shown; Figure 19D The fractal diagrams of the mAbX1 x mAbX2 clamp and ligand X are shown; and Figure 19E The fractal diagram of mAbX1 x mAbX2 2+2 tandem Fab heterodimer and ligand X is shown.

[0125] Figure 20A-20D : Figure 20A-20D This is a diagram showing the binding of antigen Y Fc-Fab to antigen Y-expressing cells, as measured in a FACS-based assay. Figure 20AAs shown, anti-antigen Y IgG1 mAb mAbY1 was cloned into IgG1 Fc-Fab (G4S disclosed as SEQ ID NO: 3) with a G4S linker of different lengths. Fc-Fab and parental IgG1 mAb exhibited similar binding to cell surface antigen Y. Figure 20B , 20C In 20D, anti-antigen Y IgG4 mAb, mAbY2, mAbY3, and mAbY4 were cloned into IgG4 Fc-Fabs with different G4S linkers (G4S is disclosed as SEQ ID NO: 3). All Fc-Fabs showed strong activity in antigen Y FACS binding assays. Figure 20A-20D The “1xG4S”, “2xG4S”, “3xG4S”, “4xG4S” and “5xG4S” disclosed in the paper are SEQ ID NO: 3, 18, 4, 19 and 39 respectively.

[0126] Figures 21A-21B : Figures 21A-21B This diagram illustrates the binding of anti-CD3 and anti-antigen Z Fc-Fab to cell surface epitopes in FACS-based assays. Anti-CD3 ( Figure 21A ) and anti-antigen Z ( Figure 21B Ab was cloned into IgG1 Fc-Fabs with G4S linkers of varying lengths (G4S is disclosed as SEQ ID NO: 3). These Fc-Fabs exhibited interaction with cell surface CD3 ( Figure 21A ) and antigen Z ( Figure 21B ) specific binding. Figures 21A-21B The “1xG4S”, “2xG4S”, “3xG4S”, “4xG4S” and “5xG4S” disclosed in the paper are SEQ ID NO: 3, 18, 4, 19 and 39 respectively.

[0127] Figures 22A-22B : Figure 22A and 22B This is a graph showing the activity of CD3 x antigen Z bispecific Fc-Fab in bioassays. Figure 22A CD3 x antigen Z bispecific Fc-Fab activates TCR signaling in Jurkat / NFAT-luciferase reporter cells in the presence of antigen Z+ cells. Figure 22B The CD3x antigen Z bispecific Fc-Fab triggered the killing of antigen Z+ cells by pre-activated human donor T cells in a 3-hour calcein release assay. Figures 22A-22B The "1xG4S", "2xG4S" and "3xG4S" disclosed in the document are SEQ ID NO: 3, 18 and 4, respectively. Detailed Implementation

[0128] 6.1. Definition

[0129] As used herein, the following terms are intended to have the following meanings:

[0130] Antibody: As used herein, the term "antibody" means any antigen-binding molecule or molecular complex comprising at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. The term "antibody" encompasses immunoglobulin molecules in their conventional form, comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and their polymers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The common amino acid sequence can be defined based on the side-by-side analysis of two or more CDRs.

[0131] As used herein, the term "antibody" also includes the antigen-binding fragment of the complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment," etc., encompass any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The antigen-binding fragment of an antibody can be derived from the complete antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding variable and optionally constant domains of the antibody. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (containing, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated by chemical methods or by using molecular biology techniques, for example, arranging one or more variable and / or constant domains into suitable conformations, or introducing codons, generating cysteine ​​residues, modifying, adding, or deleting amino acids, etc.

[0132] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues from the hypervariable region of a mimicking antibody (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, biantibodies, triantibodies, tetraantibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variant IgNAR domains, are also included in the term "antigen-binding fragments" as used herein.

[0133] Antigen-binding fragments of antibodies typically include at least one variable domain. The variable domain can have any size or amino acid composition and will typically include at least one CDR adjacent to or within one or more frame sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can contain a monomeric VH or VL domain.

[0134] In some embodiments, the antigen-binding fragment of the antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of the variable and constant domains that may be found within the antigen-binding fragment of the antibody of this disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of the variable and constant domains (including any of the exemplary configurations listed above), the variable and constant domains may be directly connected to each other or connected via complete or partial hinge or linker regions. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that result in flexible or semi-flexible connections between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of this disclosure may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above, which are non-covalently associated with each other and / or with one or more monomeric VH or VL domains (e.g., via disulfide bonds).

[0135] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically comprise at least two distinct variable domains, each capable of specifically binding to a single antigen or a different epitope on the same antigen. Conventional techniques available in the art can be used to adapt any multispecific antibody form containing the exemplary bispecific antibody forms disclosed herein to the context of antigen-binding fragments of the antibodies of this disclosure.

[0136] Antigen-binding molecules or ABM: As used herein, the term "antigen-binding molecule" or "ABM" refers to a molecule comprising two half-antibodies (e.g., an assembly of multiple polypeptide chains). Typically, each half-antibody includes at least one antigen-binding site. The ABMs of this disclosure can be monospecific or multispecific (e.g., bispecific). In monospecific binding molecules, the antigen-binding sites all bind to the same epitope, while in multispecific binding molecules, at least two antigen-binding sites bind to different epitopes, which can be the same or different target molecules.

[0137] Agreement:In the context of ABM, the term "association" refers to a functional relationship between two or more polypeptide chains. Specifically, the term "association" refers to the association of two or more polypeptides with each other, for example, non-covalent association through molecular interactions or covalent association through one or more disulfide bonds or chemical crosslinks, thereby creating a functional ABM in which antigen-binding sites can bind their respective targets. Examples of association that may exist in the ABMs of this disclosure include (but are not limited to) association between homodimeric or heterodimeric Fc domains in the Fc region, association between VH and VL regions in the Fab domain, association between CH1 and CL in the Fab domain, and association between CH3 and CH3 in domain-substituted Fab.

[0138] Bivalent: As used herein, the term "bivalent" refers to an ABM having two antigen-binding sites. In some embodiments, the two antigen-binding sites bind to the same epitope of the same target. In other embodiments, the two antigen-binding sites specifically bind to different epitopes, whether epitopes of the same target molecule or different target molecules.

[0139] Complementary Determinant Region or CDR: As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid sequence within the antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, HCDR-H3), and each light chain variable region has three CDRs (CDR1-L1, CDR-L2, CDR-L3). Exemplary specifications that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, the ABS definition, and the IMGT definition. See, for example, Kabat, 1991, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al., 1997, *Journal of Molecular Biology*, 273:927-948 (Chothia numbering scheme); Martin et al., 1989, *Proceedings of the National Academy of Sciences of the United States of America*, 86:9268-9272 (ABS numbering scheme); and Lefranc et al., 2003, *Dev. Comp. Immunol.*, 27:55-77 (IMGT numbering scheme). Public databases can also be used to identify CDR sequences within antibodies.

[0140] Cytokines: The term "cytokine" refers to a group of low-molecular-weight extracellular polypeptides / glycoproteins with cell signaling activity, including chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Cytokines are responsible for regulating immune responses (e.g., the activity, differentiation, proliferation, and production of cells and other cytokines) and are typically synthesized by immune cells, primarily T cells, neutrophils, and macrophages, but can also be synthesized by non-immune cells. Cytokines exist as monomers, dimers (homodimeters and heterodimers), trimers (including homotrimers), and tetramers (including homotetramers). The molecular weight of cytokines ranges from approximately 5 to 70 kDa, although most fall within the range of approximately 5 to approximately 20 kDa. Many cytokines have a tetra-α-helical bundle structure. Other cytokines are characterized by cysteine ​​knots containing three disulfide bonds formed by paired cysteine ​​residues. Other cytokines are characterized by a homotrimeric pyramidal structure, a feature sometimes found in cell surface proteins.

[0141] EC50: The term "EC50" refers to the half-maximal effective concentration (MC50) of an antibody or ABM, which induces half the response between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of the antibody or ABM where 50% of its maximum effect is observed. In some embodiments, the EC50 value is equal to the concentration of the antibody or ABM that produces a half-maximal binding to cells expressing a target molecule, which can be specifically bound by the antibody or ABM, for example, as determined by a FACS binding assay. Therefore, as the EC50 or half-maximal effective concentration value increases, a decrease or weakening of binding is observed. In some embodiments, the EC50 value of the ABM of this disclosure may be characterized by approximately 10... -5 M or a smaller EC50 value (e.g., less than 10) -5 M, less than 10 -6 M, less than 10 -7 M, less than 10 -8 M or less than 10 -9 M).

[0142] Epitope:The term "epitaph" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody or antigen-binding molecule, known as a complementary site. A single antigen or target molecule can have more than one epitope. Therefore, different antibody or antigen-binding molecules can bind to different regions on the antigen or target molecule and can have different biological effects. Epitopes can be conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes generated from adjacent amino acid residues in the polypeptide chain. In some cases, epitopes can contain sugar, phosphoryl, or sulfonyl groups on the antigen or target molecule.

[0143] Fab In the context of the ABM disclosed herein, the term "Fab" refers to a pair of polypeptide chains, the first comprising a variable heavy (VH) domain of the antibody located at the N-terminus of a first constant domain (referred to herein as C1), and the second comprising a variable light (VL) domain of the antibody located at the N-terminus of a second constant domain (referred to herein as C2) capable of pairing with the first constant domain. In native immunoglobulins, VH is located at the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is located at the N-terminus of the constant domain (CL) of the light chain. The Fab of this disclosure may be arranged according to the natural orientation or may include substitutions or exchanges of domains that facilitate proper VH and VL pairing, specifically in cases where the ABM of this disclosure comprises different Fabs. For example, the CH1 and CL domain pairs in the Fab may be replaced with a CH3 domain pair to facilitate proper Fab chain pairing in the heterodimeric ABM. CH1 and CL may also be reversed, such that CH1 is linked to VL and CL is linked to VH; this configuration is commonly referred to as a Crossmab. Alternatively, or in addition to using constant structural domains with substitution or exchange, proper chain pairing can be achieved by using general light chains that can pair with the two variable regions of the heterodimer ABM of this disclosure. In describing the ABM of this disclosure, the C1 structural domain is referred to elsewhere in the specification as the CH1 structural domain, and the C2 structural domain is referred to herein as the CL structural domain, for each description; however, it is also intended to include forms with exchanged domains. Section 6.2.1 illustrates other forms of engineered Fab.

[0144] FcThe term "Fc" refers to a portion of the heavy chain constant region that includes at least the CH2 and CH3 domains that normally bind to Fc receptors (e.g., FcγR, i.e., FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), or FcRn, i.e., the neonatal Fc receptor). The term "Fc" also encompasses engineered Fcs that differ from the Fc of natural immunoglobulins. For example, the CH2 and CH3 regions may be engineered to include deletions, substitutions, and / or insertions, or other modifications that prevent them from binding to any Fc receptor; in such cases, the CH2 and CH3 regions are considered non-functional in terms of their typical biological function. Section 6.2.7 illustrates other forms of engineered Fcs.

[0145] Fc structural domain and Fc region: The term "Fc domain" refers to a portion of a heavy chain that pairs with a corresponding portion of another heavy chain. The term "Fc region" refers to a region of an antibody-based binding molecule formed by the association of two heavy chain Fc domains. The two Fc domains within an Fc region may be identical or different from each other. In natural antibodies, the Fc domains are typically identical; however, for the purposes of generating the ABM of this disclosure, one or both Fc domains may be advantageously modified to allow heterodimerization.

[0146] Half antibody: The term "half-antibody" refers to a molecule that includes at least an Fc domain and can associate with another molecule including an Fc domain through, for example, disulfide bonds or molecular interactions (e.g., intrapore knob interactions between Fc heterodimers). A half-antibody can consist of one polypeptide chain or more than one polypeptide chain (e.g., heavy and light chains).

[0147] Heavy chain: As used herein, the term "heavy chain" or "immunoglobulin (Ig) heavy chain" includes the Ig heavy chain constant region sequence from any organism and, unless otherwise stated, includes heavy chain variable domains. Unless otherwise stated, a heavy chain variable domain contains three heavy chain complementarity-determining regions (CDRs) and four framework regions (FRs). A fragment of a heavy chain variable domain contains either a CDR or both CDRs and FRs. A typical heavy chain constant region (CH) following the variable domain, from the N-terminus to the C-terminus, has: a CH1 domain, a hinge, a CH2 domain, and a CH3 domain (see example...). Figure 1A and 2A Atypical heavy chains, such as those disclosed in this paper concerning antigen-binding molecules and bispecific reantigen-binding molecules, have variable domains (VH) between any two constant regions (CH) of the heavy chain, for example, from the N-terminus to the C-terminus: CH2 domain, CH3 domain, VH domain, and CH2 domain (see example). Figure 1B and Figure 2BIn one embodiment, the Fc portion includes at least the CH2 and CH3 domains.

[0148] Hinge: As used herein, the term "hinge" is intended to encompass the region of consecutive amino acid residues that link the C-terminus of the CH1 domain of an immunoglobulin to the N-terminus of the CH2 domain. Several amino acids encoded by the CH2 exon at the N-terminus of the CH2 domain are also considered part of the "lower hinge." Unbound by any one theory, the amino acids in the hinge regions of IgG1, IgG2, and IgG4 have been characterized as comprising 12–15 consecutive amino acids encoded by different hinge exons, and several N-terminal amino acids of the CH2 domain (encoded by the CH2 exon) (Brekke et al., 1995, Immunology Today 16(2):85–90). On the other hand, IgG3 comprises a hinge region consisting of four segments: an upper segment similar to that of IgG1, and three segments that are identical amino acid repeats unique to IgG3.

[0149] Host cell: As used herein, the term "host cell" refers to a cell in which nucleic acids of this disclosure have been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms refer to a specific subject cell and its progeny or potential progeny. Such progeny may differ in fact from the parent cell due to mutations or environmental influences that may result in certain modifications in the offspring, but are still included within the scope of the terminology used herein. Typical host cells are eukaryotic host cells, such as mammalian host cells. Exemplary eukaryotic host cells include yeast and mammalian cells, such as vertebrate cells, such as mouse, rat, monkey, or human cell lines, such as HKB11 cells, PER.C6 cells, HEK cells, or CHO cells.

[0150] Immunoglobulins: The term "immunoglobulin" (Ig) refers to a class of structure-associated glycoproteins composed of two pairs of polypeptide chains, one pair of light (L) chains, and one pair of heavy (H) chains, all four pairs linked together by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Chapter 7 of *Fundamental Immunology* (edited by Paul, W., 2nd ed., Raven Press, New York (1989)). Each heavy chain typically includes a variable region (abbreviated as VH or VH in this text) and a constant region (CH or CH). The constant region typically includes three domains: CH1, CH2, and CH3. The CH1 and CH2 domains are linked by hinges. The Fc portion includes at least the CH2 and CH3 domains.

[0151] Typically, the amino acid residue numbering of immunoglobulins is based on IMGT, “Protein Sequences of Immunological Significance,” 5th edition, Public Health Service, NIH, Bethesda, Maryland (1991), or by Kabat’s EU numbering system (also known as “EU Numbering” or “EU Index”), as described in, for example, Kabat et al., “Protein Sequences of Immunological Significance,” 5th edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991).

[0152] Same type: The term "isotype" refers to an immunoglobulin class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by genes in the heavy chain constant region.

[0153] Operable connection The term "operably linked" refers to the physical or functional juxtaposition of components so described that allows them to function in their intended manner. In the context of polypeptides, "operably linked" can refer to a functional relationship between two or more regions of a polypeptide chain, wherein the two or more regions are linked to produce a functional polypeptide. In the context of nucleic acids, such as in the context of DNA expression vector constructs, the term "operably linked" refers to, for example, a control sequence, such as a promoter or operon, being appropriately placed relative to a coding sequence such that the control sequence directs the production of a polypeptide encoded by the coding sequence.

[0154] Polypeptides and proteins: The term "protein" is intended to include quaternary, tertiary, and other complex macromolecules composed of at least one polypeptide. The term "protein" includes polypeptides.

[0155] The term "peptide" refers to a linear polymer chain of single amino acids linked together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Peptides disclosed herein include amino acid sequences derived from immunoglobulin domains. "Derived from" refers to the peptide or amino acid sequence of a protein or polypeptide, indicating the source of the peptide.

[0156] The term "protein" can also be used to describe large polypeptides, such as polypeptides composed of one or more polypeptides.

[0157] Single-chain Fab: As used herein, the term “single-chain Fab” or “scFab” refers to a polypeptide chain that includes the VH, CH1, VL, and CL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0158] Single-chain Fv or scFv:As used herein, the term "single-chain Fv" or "scFv" refers to a polypeptide chain that includes the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.

[0159] Specific (or selective) binding: As used herein, the term "specific (or selective) binding" refers to the formation of a complex between ABM or its antigen-binding site ("ABS") and a target molecule that is relatively stable under physiological conditions. Specific binding is characterized by approximately 5 x 10⁻⁶ ppm. -2 M or smaller KD (e.g., less than 5x10) -2 M, less than 10 -2 M, less than 5x10 -3 M, less than 10 - 3 M, less than 5x10 -4 M, less than 10 -4 M, less than 5x10 -5 M, less than 10 -5 M, less than 5x10 -6 M, less than 10 -6 M, less than 5x10 -7 M, less than 10 -7 M, less than 5x10 -8 M, less than 10 -8 M, less than 5x10 -9 M, less than 10 -9 M or less than 10 -10 Methods for determining the binding affinity of an antibody or antibody fragment (e.g., ABM or ABS) to a target molecule are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., Biacore assay), fluorescence activated cell sorting (FACS) binding assay, etc. However, ABM or ABS antibodies that specifically bind to target molecules from one species may be cross-reactive with target molecules from one or more other species.

[0160] Target molecule:As used herein, the term "target molecule" refers to any biomolecule (e.g., protein, carbohydrate, lipid, or combination thereof) that can be specifically bound to the antigen-binding site of ABM. Exemplary target molecules include, but are not limited to, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRLI, ADORA2A, agglutinin, AGR2, AICDA, AIF1, AIG1, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, AZGP1 (zinc-α-glycoprotein), ART-4, B7, B7.1, B7.2, BAD, BAFF, BAGI, BAIi, BCL2, BCL6, BDNF, BLNK, and BLR. l (MDRlS), BlyS, BMPl, BMP2, BMP3B (GDF10), BMP4, BMP6, BMPS, BMPR1A, BMPR1B, BMPR2, BPAG1 (lectin), BRCA1, Ba-733, BAGE, BrE3-antigen, CA125, CA MEL, CAP-I, CASP-8 / m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CDS, CD8, CDI-IA, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD2 5. CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95 , CD126, CD133, CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CXCR4, CXCR7, CXCL12, C19orf10 (IL27w), C3, C4A, CS, CSR1, CANT1, CASPI, CAS P4, CAV1, CCBP2 (D6 / JAB61), CCLI (I-309), CCLII (chemokine), CCL13 (MCP-4), CCLIS (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCLIS (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MIP-2), SLC, exodus-2, CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / chemokine-2)CCL2S (TECK), CCL26 (chemokine-3), CCL27 (CTACK / ILC), CCL2S, CCL3 (MIP1a), CCL4 (MIP-1b), CCLS (RANTES), CCL7 (MCP-3), CCLS (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1 / HM14S), CCR2 (mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCRS (CMKBRSI) ChemR13) CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EB1), CCRS (CMKBRS / TER1 / CKR-LI), CCR9 (GPR-9-6), CCRLI (VSH K1), CCRL2 (L-CCR), CD164, CDlC, CD200, CD-22, CD24, CD2S, CD3S, CD3E, CD3G, CD3Z, CD4, CD44, CD4SRB, CD47, CD4S, CDS2, CD 69. CD72, CD79A, CD79B, CDSO, CDS1, CDS3, CDS6, CD137, CD13S, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CDH1 (E-cadherin White), CDH10, CDH12, CDH13, CDH1S, CDH19, CDH20, CDHS, CDH7, CDHS, CDH9, CDK2, CDK3, CDK4, CDKS, CDK6, CDK7, CDK9, CDKN1A (p21 Wap1 / Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEBPB, CER1, CHGA, CHGB, chitinase, CHST1O, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSFS, CKLFSF6, CKLFSF7, CKLFSFS, CLDN3, CLDN7 (blocking agent-7), CLN 3. CLU (cluster protein), CMKLR1, CMKOR1 (RDC1), CNR1, COLISA1, COLIA1, COL4A3, COL6A1, CR2, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA-4, CTNNB1 (β-catenin), CTSB (cathepsin B), CX3CLI (SCYD1), CX3CR1 (V2S), CXCLI (GRO1)CXCLIO (IP-10), CXCL11 (I-TAC / IP-9), CXCL13, CXCL14, CXCL16, CXCL2 (GR02), CXCL3 (GR03), CXCLS (ENA-7S / LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), CYBS, CYC1, CYSLTR1, HIF-1-a, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-met, DAB2IP, DES, D KFZp4S1J011S, DNCLI, DPP4, DAM, EGFR, EGFRvllll, EGP-1, EGP-2, ELF2-M, Ep-CAM, E2F1, ECGF1, EDG1, EFNA1, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, EN 01, EN02, EN03, EPHB4, EPO, EREG, ERKS, ESR1, ESR2, F3 (TF), FADD, FasL, FASN, FCER1A, FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF11, FGF12, FGF12B FGF13, FGF14, FGF16, FGF17, FGF1S, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGFS, FGF7 (KGF), FGFS, FGF9, FGFR3, FIGF (VEGFD), FILI (EPSILON), FILI (ZETA), FLJ12SS4, FLJ2SS30, FLRT1 (fibronectin), FOS, FOSLI (FRA-1), FY (DARC), Flt-I, Flt-3, folic acid receptor, G250 antigen, GAGE. GROB, GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GDFS, GFil, GGT1, GM-CSF, GNAS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPRS1 (FKSGSO), GRCC10 (C10), GRP, GSN (gelatin), GSTP1, HAVCR2, HDAC4, HDACS, HDAC7A, HDAC9, HGF, HIP1 histamine and histamine receptor, HLA-A, HLA-DRA, HM74, HMOX1, HUMCYT2A, HLA-DR, HMI 24Human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2 or 1a, IGF-IR, IFN-ɣ, IFN-α, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IGBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-1, IL-10, IL-10RA, IL-10RB, IL-11, IL-11RA, IL-12, IL-12A, IL-12B, IL-12RB_{1}, IL-12RB_{2}, IL-13, IL-13RA_{1}, IL-13RA_{2}, IL-14, IL-1S, IL-1SRA, IL-16, IL-17, IL-17B, IL-17C, IL-17R, IL-18, IL-18BP, IL-18R_{1}, IL-18RAP, IL-19, IL-IA, IL-1B, IL-1F10, IL-1FS, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-1HY1, IL-1R_{1}, IL-1R_{2}, IL-1RAP, IL-1RAPL_{1}, IL-1RAPL_{2}, IL-1RL_{1}, IL-1RL_{2} IL-1RN, IL-2, IL-20, IL-20RA, IL-21R, IL-22, IL-22R, IL-22RA_{2}, IL-23, IL-24, IL-2S, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-2RA, IL-2RB, IL-2RG, IL-3, IL-30, IL-3RA, IL-4, IL-4R, IL-S, IL-5RA, IL-6, IL-6R, IL-6ST (glycoprotein 130), IL-7, IL-7R, IL-S, IL-SRA, IL-SRB, IL-9, IL-9R, IL-K, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA_{1}, ITGA_{2}, ITGA_{3}, ITGA_{6} (α6 integrin), ITGAV, ITGB_{3}, ITGB_{4} (β4 integrin), insulin-like growth factor-I (IGF-1), ICEBERG, ICOS-L, ID2, IFN-α, IFNA_{1}, IFNA_{2}, IFNA_{4}, IFNAS, IFNA_{6}, IFNA_{7}, IFNB_{1}, IFNW_{1}, JAG1, JAK1, JAK3, JUN, K6HF, KAI1, KDR, KITLG, KLF5 (GC box-binding protein),KLF6, KLK10, KLK12, KLK13, KLK14, KLK1S, KLK3, KLK4, KLKS, KLK6, KLK9, KRT1, KRT19 (keratin 19), KRT2A, KRTHB6 (hair-specific type II keratin), KC4-antigen, KS-1-antigen, KS 1-4, Le-Y, LDR / FUT, LAMAS, LEP (leptin), Lingo-p7S, Lingo-Troy, LPS, LTA (TNF-β), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​MACMARCKS, MAG or Omgp, MAP2K7 (c-Jun), MDK, MIB1, intermediate factor, MIF, MIP-2, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionein-III), MTSS1, MUC1 (mucin) MYC, MYD88, Macrophage Migration Inhibitor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, NCK2, Neuroenergies, NFKB1, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgR-Nogo66 (Noga) NgRp7S, NgR-Troy, NME1 (NM23A), NOXS, NPPB, NROB1, NROB2, NR1D1, NR1D2, NRIH2, NRIH3, NRIH4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E1 , NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NRSA1, NRSA2, NR6A1, NRP1, NRP2, NTSE, NTN4, ODZ1, OPRD1, PCSK9 P2RX7, PAP, PART1, PATE, PAWR, PCA3, PCNA, PD-1, PD-L1, α4β7, OX40, GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDFS, CGRP, Lingo-I, Factor IXa, Factor X, ICOS, GARP, BTLA, CD160, RORI, 2B4, KIR, CD27, OX40, A2aR, PDGFA, PDGFB, PECAM1, PF4 (CXCL4), PGF, PGR, phosphatidylcholine, PIAS2, PIK3CG,PLAU (uPA), PLG, PLXDC1, PPBP (CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2 (COX-2), PTN, pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, 10 PIGF, ILGF, ILGF-IR, IL-6, RS5, RANTES, RAC2 (p21Rac2), RARB, RGS1, RGS13, RGS3, RNF110 (ZNF144), ROB02, S100A2, SCGB1D2 (lipophilic B), SCGB2A1 (mammary globin 2), SCGB2A2 (mammary globin 1), SCYE1 (endothelial monocyte activation cytokine), SDF2, SERPINA1, SERPINA3, SERPINABS (mammary filament inhibitory protein), SERPIN E1 (PAI-1), SERPINF1, SHBG, SLA2, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPP1, SPRR1B (Sprl), ST6GAL1, STAB1, STATE, STEAP, STEAP2, TIOI, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tendinogen, TRAIL receptor, TNF-α, Tn-antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, TB4R2, TBX2 1. TCP10, TDGF1, TEK, TGFA, TGFB1, TGFBlil, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, THBS1 (platelet-reactive protein-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor, TLR10, TLR2, TLR3, TLR4, TLRS, TLR6, TLR7, TLR8, TLR9, TNF, TNF-α, TNFAIP2 (B94), TNF AIP3, TNFRSF11A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSFS, TNFRSF6 (Fas), TNFRSF7, TNFRSFS, TNFRSF9, TNFSF10 (TRAIL), TNF SF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF1S (VEGI), TNFSF18, TNFSF4 (OX40 ligand),TNFSFS (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSFS (CD30 ligand), TNFSF9 (4-lBB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TPS3, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAPS, TRAF6, TREM1, TREM2, TRPC6, TSLP, TWEAK, VEGFR, ED-B fibronectin, WT-1, 17-IA antigen, complement factor C3, C3a, C3 b. C5a, CS, angiogenesis markers, bcl-2, bcl-6, Kras, cMET, CD19 / CD3, BCMA / CD3, EGFR, HER3, IL17RA / IL7R, IL-6 / IL-23, IL1 / IL-8, IL-6, IL-6R / IL-21, IL-21R, ANG2 / VEGF, VEGF / PDGFR-β, vascular endothelial growth factor (VEGF) receptor 2 / CD3, PSMA / CD3, EPCAM / CD3, VEGFR-1, VEGFR-2, VEGFR-3, VEGFB, VEGFC, multifunctional proteoglycans, VHL CS, VLA-4, c-FMS / CSFIR, RET, HER3, HER4, IGFR, PDGFR, c-KIT, BCR, integrins, MMPs, VEGF, EGF, PIGF, PDGF, HGF, angiopoietin, ERBB-3 / C-MET, ERBB-2 / C-MET, EGF receptor 1 / CD3, EGFR / HER3, PSCA / CD3, C-MET / CD3, ENDOSIALIN / CD3, EPCAM / CD3, IGF-1R / CD3, FAPALPHA / CD3, EGFR / IGF-IR, IL 25 17A / F, EGF receptor 1 / CD3 and CD19 / CD16, KHI, Tn-antigen, TF-antigen, CD44, glycolipids, glycosphingolipids, such as 30 Gg3, Gb3, GD3, GD2, Gb5, Gm1, Gm2, sialytetrasaccharide ceramide, XCL1 (lymphocyte chemokine), XCL2 (SCM-1b), XCR1 (GPRS / CCXCR1), YY1, and ZFPM2. In some embodiments, the target molecule is a small, soluble (i.e., non-membrane-bound) molecule.

[0161] Quadrivalent:As used herein, the term "tetravalent" refers to an ABM having four antigen-binding sites. In some embodiments, two of the antigen-binding sites bind to the same epitope, while the other two binding sites bind to different epitopes, whether epitopes of the same or different target molecules.

[0162] General heavy chain: As used herein in the context of ABM, the term "universal heavy chain" refers to a heavy chain with rearranged heavy chain variable regions, such as a human heavy chain with rearranged Ig heavy chain variable regions. Exemplary rearranged Ig heavy chain variable regions are provided in U.S. Patent Publication No. 2014 / 0245468 and U.S. Patent Nos. 9,204,624 and 9,930,871, each of which is incorporated herein by reference in its entirety. Universal heavy chains are also referred to as "common heavy chains".

[0163] General-purpose light chain: As used herein in the context of ABM, the term "universal light chain" refers to a light chain having rearranged light chain variable regions, such as a human heavy chain having rearranged Ig light chain variable regions. Universal light chains are also referred to as "common heavy chains." In the context of ABM, it refers to a light chain polypeptide capable of pairing with heavy chain regions of two different Fab domains having different variable regions in the same ABM. Universal light chains are also referred to as "common light chains." Exemplary rearranged Ig light chain variable regions are provided, for example, in U.S. Patent Nos. 9,969,814, 10,130,181, and 10,143,186, and U.S. Patent Publications 2012 / 0021409, 2012 / 0192300, 2013 / 0045492, 2013 / 0185821, 2013 / 0302836, and 2015 / 0313193, each of which is incorporated herein by reference in its entirety.

[0164] VH: The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody, which contains the Fab heavy chain.

[0165] VL: The term "VL" refers to the variable region of the immunoglobulin light chain, which contains the Fab light chain.

[0166] 6.2. Antigen-binding molecules (ABM)

[0167] This paper discloses antigen-binding molecules, such as monospecific and bispecific antigen-binding molecules. The disclosed antigen-binding molecules have binding domain arrangements that differ from typical antibody architectures. The disclosed antigen-binding molecules can bispecifically bind to a single target molecule or antigen, which may result in increased affinity and / or cohesion to the antigen or target molecule. For example, for a bispecific antigen-binding molecule in which two Fab domains bind the same antigen at different epitopes, increased affinity for the antigen is expected compared to an antibody that binds only one epitope. Without being bound by theory, it is believed that the ABMs disclosed herein have increased affinity for antigens or target molecules due to increased proximity and / or greater flexibility of the Fab1 and Fab2 domains, which will increase the local concentration of the antigen-binding site compared to conventional antibody forms where the binding sites of the Fab domains are spaced apart.

[0168] In a first aspect, the ABM disclosed herein includes:

[0169] ●The first half-antibody, wherein the first half-antibody includes the following orientation from the N to the C terminus:

[0170] -Optional hinge structure domain;

[0171] - The first Fc structural domain; and

[0172] - A first Fab (Fab1) structural domain, the first Fab structural domain including a first heavy chain variable region (VH) associated with a first light chain variable region (VL); and

[0173] ●The second half-antibody, wherein the N-to-C-terminal orientation of the second half-antibody includes:

[0174] -Optional hinge structure domain;

[0175] - The second Fc structural domain; and

[0176] - A second Fab (“Fab2”) domain, the second Fab domain including a second VH associated with a second VL.

[0177] The first Fc structural domain and the second Fc structural domain associate with each other to form an Fc region, and the optional hinge structural domain, if present, can associate with each other via disulfide bonds.

[0178] Figure 1B and Figure 2B Two embodiments of this type of ABM are shown herein, which are generally referred to herein as ABM form "A" ("Type A") and sometimes as "Fc-Fab" form, and are depicted in Figure 13A , Figure 13B and Figure 13C Variations thereof. Therefore, this disclosure provides depictions of... Figure 1B and Figure 2B Type A ABM includes:

[0179] ● First polypeptide, the first polypeptide comprising, in the N-to-C-terminal orientation:

[0180] - Optional hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond;

[0181] -Fc domain, the Fc domain includes CH2 domain (4) and CH3 domain (5).

[0182] - Optional hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond;

[0183] - Connector (8); and

[0184] -The heavy chain component of the Fab1 domain, comprising the Fab1 VH domain (1) and the Fab1 CH1 domain (2) associated with the light chain component of the Fab1 domain, wherein the light chain component in polypeptide form comprises the Fab1 VL domain (6) and the Fab1 CL domain (7) oriented from N to C; and

[0185] ●The second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0186] - Optional hinge domain (3), which is connected to the hinge domain in the first polypeptide via a disulfide bond;

[0187] - Second Fc domain, which includes CH2 domain (4) and CH3 domain (5).

[0188] - Optional hinge domain (3), which is connected to the hinge domain in the first polypeptide via a disulfide bond;

[0189] - Connector (8); and

[0190] -The heavy chain component of the Fab2 domain includes the Fab2 VH domain (1) and the Fab2 CH1 domain (2) associated with the light chain component of the Fab2 domain, and the light chain component in polypeptide form includes the Fab2 VL domain (6) and the Fab2 CL domain (7) in the N-to-C-terminal orientation.

[0191] The first Fc structural domain and the second Fc structural domain associate with each other to form the Fc region.

[0192] exist Figure 1BIn the embodiments described, the two half-antibodies are identical, containing Fc domains that form Fc homodimers, and the resulting ABM is single-specific. Figure 2B In some embodiments, ABM includes Fc heterodimers, thereby allowing the use of different Fab1 and Fab2 VH domains and producing multispecific, such as bispecific, molecules. Although Figure 1B , Figure 2B and 13A An embodiment of the ABM is shown, in which a hinge region is formed by a hinge structure domain at the N end of the Fc structure domain. However, type A ABMs may not have a hinge region (not shown) or may have a hinge region at the C end of the Fc region. Figure 13C ) or a hinge area with N and C ends of the Fc region ( Figure 13B ).like Figures 13A-13C The exemplary hinge domains depicted that can be used at the N-terminus and / or C-terminus of the Fc region include the amino acid sequences GGGGSCPPC (SEQ ID NO: 1) and ESKYGPPCPPC (SEQ ID NO: 2), but type A ABM can also have alternative hinge region sequences. Similarly, although... Figures 13A-13C It depicts (G4S). n The connector (G4S is disclosed as SEQ ID NO: 3) can be used, but other connector sequences may be used.

[0193] Although Figure 1B and Figure 2B An embodiment of a type A ABM containing only two bonding domains (Fab1 and Fab2) is shown, but the ABM of this disclosure may contain additional bonding domains, such as scFv or Fab domains. However, in some respects, Fab1 and Fab2 are the only bonding domains of the type A ABM.

[0194] In a second aspect, the ABM disclosed herein includes:

[0195] ●The first half-antibody, wherein the first half-antibody includes the following orientation from the N to the C terminus:

[0196] - A first Fab (Fab1) structural domain, the first Fab structural domain including a first VH associated with a first VL;

[0197] - First spacer structural domain; and

[0198] - The first Fc structural domain; and

[0199] ●The second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0200] - A second Fab (Fab2) structural domain, the second Fab structural domain including a second VH associated with a second VL;

[0201] - Second spacer structure domain; and

[0202] -Second Fc structural domain; and

[0203] The first Fc structural domain and the second Fc structural domain associate with each other to form the Fc region.

[0204] Unbound by theory, it is believed that including a spacer domain between the Fc and Fab domains would result in greater flexibility between the antigen-binding sites of the Fc and Fab regions, and thus produce higher affinity and / or cohesion of ABM to its target molecules.

[0205] In some embodiments, the spacer structure domain is an extension joint. Figure 3A This ABM form is illustrated herein and is generally referred to herein as form "B" ("Type B") and sometimes as the "extended form". Therefore, this disclosure provides a depiction of... Figure 3A An embodiment of type B ABM includes:

[0206] ● First polypeptide, the first polypeptide comprising, in the N-to-C-terminal orientation:

[0207] -The heavy chain component of the Fab1 domain includes the Fab1 VH domain (1) and the Fab1 CH1 domain (2) associated with the light chain component of the Fab1 domain, and the light chain component in polypeptide form includes the Fab1 VL domain (6) and the Fab1 CL domain (7) in the N-to-C-terminal orientation.

[0208] - Connector structural domain (8), the connector structural domain being an extension connector;

[0209] - Hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond; and

[0210] - A first Fc domain, comprising a CH2 domain (4) and a CH3 domain (5); and

[0211] ●The second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0212] -The heavy chain component of the Fab2 domain includes the Fab2 VH domain (1) and the Fab2 CH1 domain (2) associated with the light chain component of the Fab2 domain, and the light chain component in polypeptide form includes the Fab2 VL domain (6) and the Fab2 CL domain (7) in the N-to-C-terminal orientation.

[0213] - Connector structural domain (8), the connector structural domain being an extension connector;

[0214] - Hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond; and

[0215] - Second Fc domain, which includes CH2 domain (4) and CH3 domain (5).

[0216] Although Figure 3A The embodiment of the B-type ABM depicted contains only two bonding domains (Fab1 and Fab2), but the B-type ABM of this disclosure may contain additional bonding domains, such as scFv or Fab domains. However, in some respects, Fab1 and Fab2 are the only bonding domains of the B-type ABM of this disclosure.

[0217] In other embodiments, the spacer domain is a Fab domain. Figure 3B-3D Different variants of this ABM form are shown in the document, referred to herein as form "C" ("C-type"). Therefore, the C-type ABM comprises a third Fab (Fab3) structural domain and a fourth Fab (Fab4) structural domain, with the following configuration:

[0218] ●The first hemibody, wherein the first hemibody includes, in the N-to-C-terminal orientation...

[0219] - A first Fab (Fab1) structural domain, the first Fab structural domain including a first VH associated with a first VL;

[0220] - A third Fab (Fab3) structural domain, the third Fab structural domain including a third VH associated with a third VL; and

[0221] - The first Fc structural domain; and

[0222] ●The second half-antibody, wherein the N-to-C-terminal orientation of the second half-antibody includes:

[0223] - A second Fab (Fab2) structural domain, the second Fab structural domain including a second VH associated with a second VL;

[0224] - A fourth Fab (Fab4) structural domain, the fourth Fab structural domain including a fourth VH associated with a fourth VL; and

[0225] -Second Fc structural domain.

[0226] Therefore, this disclosure provides a description of... Figure 3B-3D An embodiment of type C ABM includes:

[0227] ● First polypeptide, the first polypeptide comprising, in the N-to-C-terminal orientation:

[0228] -The heavy chain component of the Fab1 domain includes the Fab1 VH domain (1) and the Fab1 CH1 domain (2) associated with the light chain component of the Fab1 domain, and the light chain component in polypeptide form includes the Fab1 VL domain (6) and the Fab1 CL domain (7) in the N-to-C-terminal orientation.

[0229] - Connector structural domain (8);

[0230] -The heavy chain component of the Fab3 domain includes the Fab3 VH domain (1) and the Fab3 CH1 domain (2) associated with the light chain component of the Fab3 domain, and the light chain component in polypeptide form includes the Fab3 VL domain (6) and the Fab3 CL domain (7) in the N-to-C-terminal orientation.

[0231] - Hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond; and

[0232] - A first Fc domain, comprising a CH2 domain (4) and a CH3 domain (5); and

[0233] ●The second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0234] -The heavy chain component of the Fab2 domain includes the Fab2 VH domain (1) and the Fab2 CH1 domain (2) associated with the light chain component of the Fab2 domain, and the light chain component in polypeptide form includes the Fab2 VL domain (6) and the Fab2 CL domain (7) in the N-to-C-terminal orientation.

[0235] - Connector structural domain (8);

[0236] -The heavy chain component of the Fab4 domain includes the Fab4 VH domain (1) and the Fab4 CH1 domain (2) associated with the light chain component of the Fab4 domain, and the light chain component in polypeptide form includes the Fab4 VL domain (6) and the Fab4 CL domain (7) in the N-to-C-terminal orientation.

[0237] - Hinge domain (3), which is connected to the hinge domain in the second polypeptide via a disulfide bond; and

[0238] - Second Fc domain, the second Fc domain includes CH2 domain (4) and CH3 domain (5); wherein the first Fc domain and the second Fc domain associate with each other to form an Fc region.

[0239] Although Figure 3B-3DThe embodiments of the C-type ABM depicted contain four bonding domains (Fab1, Fab2, Fab3, and Fab4), but the C-type ABM of this disclosure may contain additional bonding domains, such as scFv or Fab domains. However, in some respects, Fab1, Fab2, Fab3, and Fab4 are the only bonding domains of the C-type ABM of this disclosure.

[0240] The Fab3 and Fab4 domains of a C-type ABM can be non-bonded (e.g., Figure 3B (as shown) or combined (such as) Figure 3C and Figure 3D (As shown). Those embodiments where Fab3 and Fab4 are non-jointed are generally referred to herein as type C1 ABMs, and this form is sometimes referred to herein as a "clamp" form. Those embodiments where Fab3 and Fab4 are joined are generally referred to herein as type C2 ABMs, and this form is sometimes referred to herein as a "tandem Fab" form. The term "2+2 tandem Fab" refers to... Figure 3C and Figure 3D In the illustrated embodiment, Fab1, Fab2, Fab3, and Fab4 are the only binding structural domains in the tandem Fabs. Each of the C1-type and C2-type ABMs can be either a homodimer or a heterodimer.

[0241] In some embodiments of C1-type ABM, the Fab1 and Fab2 domains are different (e.g., binding different epitopes, whether on the same target molecule or different target molecules), and the Fab3 and Fab4 domains are the same non-binding domains. In other embodiments, the Fab3 and Fab4 domains are different non-binding domains.

[0242] In some embodiments of the C2 type ABM, the Fab1 and Fab3 structural domains include the same VH structural domain, and the Fab2 and Fab4 structural domains include the same VH structural domain, such as... Figure 3C As shown. This configuration is referred to as configuration 1, or 1-1-2-2 configuration. In an alternative embodiment of the C2 type ABM, the Fab1 and Fab2 domains include the same VH domain, and the Fab3 and Fab4 domains include the same VH domain, as shown. Figure 3D As shown. This configuration is called configuration 2, or 1-2-1-2 configuration.

[0243] A complete ABM is formed by the association of two half-antibodies through two Fc domains to form an Fc region. When the two half-antibodies are not identical, for example when Fab1 and Fab2 contain different VH domains, Fc heterodimerization methods (e.g., as described in Section 6.2.7.2) can be used to facilitate correct half-antibody pairing or their purification. Examples of heterodimerization methods are star mutations (as described in Section 6.2.7.2) or intrawell toggle mutations.

[0244] Although Figure 2B , 3A Figures 3B and 3C illustrate ABMs comprising distinct VH domains in each half-antibody paired via Fc heterodimers, but this form can also be used for Fc homodimers. For example, although Figure 2B and Figure 3A Examples of type A and type B ABMs are shown, each incorporating an Fc heterodimer, which allows for the incorporation of different VH domains in Fab1 and Fab2 to generate multispecific (e.g., bispecific) binding molecules. However, this form can also be used for single-specific type A and type B ABMs having an Fc homodimer and the same VH domains. Similarly, an Fc homodimer can be used to generate single-specific type C ABMs having the same Fab1, Fab2, Fab3, and Fab4 VH domains, or the same Fab1 and Fab2 VH domains and non-binding Fab3 and Fab4 VH domains.

[0245] Furthermore, when the first and second peptides contain different VH domains, different strategies can be used to allow correct VH-VL pairing in multispecific binding molecules. For example, a common light chain capable of effectively pairing with more than one type of VH domain in an ABM can be used. In such embodiments, the light chain peptides (e.g., light chains associated with Fab1 and Fab2 and, if present, Fab3 and Fab4) can be identical. Alternatively, a single-domain Fab can be used, where the heavy chain components ((1) and (2)) can be expressed fused with the light chain components ((6) and (7)).

[0246] The variations of the ABM of this disclosure shown in Figures 1-3 are not intended to be limiting; except as otherwise provided, the ABM of this disclosure may comprise any combination of the modifications shown in Figures 1-3 and Section 6.2 below. Furthermore, references to the first or second polypeptide chain or the left or right hemiantibody are for convenience only and are not intended to convey that the polypeptide chains or hemiantibodies are generated or assembled in any particular order.

[0247] In some embodiments, the first Fab (Fab1) domain and the second Fab (Fab2) domain of the ABM disclosed herein can each bind the same target molecule, such as a small soluble molecule. The first Fab (Fab1) domain and the second Fab (Fab2) domain can bind the same epitope (e.g., in...). Figure 1B and Figure 3D In the embodiments depicted, or in Figure 3A or Figure 3B In variants where Fab1 and Fab2 both have the same VH domain (not shown) or they can combine different epitopes (e.g., in... Figure 2B , Figure 3A , Figure 3B and Figure 3C In the embodiments depicted, whether on the same target molecule or on different target molecules. In the case where the first Fab (Fab1) domain and the second Fab (Fab2) domain bind different epitopes (e.g., two different epitopes on the same target molecule or on different target molecules), they can be selected so that the Fab can bind to its epitopes simultaneously.

[0248] In some embodiments, such as in a C-type ABM, the ABM of this disclosure may include a third Fab (Fab3) structural domain and a fourth Fab (Fab4) structural domain, such as... Figure 3C , Figure 3A and Figure 3D As described. The third and fourth Fab domains can be non-bonded, such as Figure 3C The epitopes described herein, or those that may be combined with the same or different epitopes as those combined by the first and second Fab (Fab1 and Fab2) structural domains respectively. As used herein, with respect to C-type ABM, the terms "first and second Fab structural domains" and "Fab1 and Fab2 structural domains" generally refer to the N-most Fab structural domain.

[0249] Certain target molecules, specifically those with repetitive epitopes, such as those found in polypeptides with repetitive motifs or proteins with multimeric structures (e.g., homodimers or homotrimers), can be bound by two or more antibody molecules, leading to the formation of large complexes. The production of large heterogeneous antibody complexes is referred to as “paper-dolling.” Large antibody complexes can be rapidly eliminated by phagocytosis, resulting in reduced antibody efficacy. Large complexes can also increase the immunogenicity of therapeutic antibodies. See, for example, WO 2020047067A1. The ABMs of this disclosure may be less prone to aggregation, e.g., in vivo or in vitro, compared to parental antibodies from which Fab domains are derived. By way of non-limiting examples, for bispecific ABMs in which two Fab domains bind the same antigen at different epitopes, it was observed (see Example 4 below) that, unlike results obtained using combinations of parental mAbs, the ABMs of this disclosure primarily form discrete 1:1 complexes with the ligand, with little or no additional higher-order complexes. In contrast, the parental mAbs combined to form multiple higher-order structures (multimers), suggesting that these parental antibodies form bridges between multiple ligands, such as forming unfolded "paper doll" structures. These results indicate that the ABMs disclosed in this paper are not prone to aggregation because the proximity of the Fab domains is believed to favor the formation of 1:1 Fc-Fab ligand complexes rather than higher-order structures. In practice, this could lead to a higher relative concentration of the individual ABM:target molecule complex than the expected concentration of the parental antibody.

[0250] In some embodiments, the ABM of this disclosure specifically binds to at least two different epitopes (and in some cases, three or four different epitopes). The at least two different epitopes may be on the same target molecule or different target molecules.

[0251] 6.2.1. Fab Domain

[0252] The ABM of this disclosure includes at least one Fab domain in each half-antibody. Fab domains are conventionally generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the ABM of this disclosure, the Fab domain is recombinantly expressed as part of a larger molecule.

[0253] The Fab domain can include constant and variable domain sequences from any suitable species, and therefore can be mouse, chimeric, human, or humanized.

[0254] The Fab domain typically includes a CH1 domain attached to the VH domain, which pairs with a CL domain attached to the VL domain. In wild-type immunoglobulins, the VH and VL domains pair to form the Fv region, and the CH1 and CL domains pair to further stabilize the binding module. A disulfide bond between the two constant domains can further stabilize the Fab domain.

[0255] Specifically, for the ABM of this disclosure, when the light chains are not common or generic light chains, it is advantageous to use a Fab heterodimerization strategy to allow the correct association of Fab domains belonging to the same ABS and to minimize anomalous pairings of Fab domains belonging to different ABS. For example, the Fab heterodimerization strategy shown in Table B below can be used:

[0256]

[0257] Therefore, in some embodiments, proper association between two peptides of Fab is facilitated by exchanging the VL and VH domains of Fab with each other or by exchanging the CH1 and CL domains with each other, for example, as described in WO 2009 / 080251.

[0258] Proper Fab pairing can also be facilitated by introducing one or more amino acid modifications into the CH1 domain and the CL domain of the Fab, and / or by introducing one or more amino acid modifications into the VH domain and the VL domain. The modified amino acids are typically part of the VH:VL and CH1:CL interfaces, causing Fab components to preferentially pair with each other rather than with components from other Fabs.

[0259] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of variable (VH, VL) and constant (CH1, CL) domains as indicated by Kabat residue numbering. Almagro, 2008, Frontiers in Bioscience 13:1619-1633 provides framework residue definitions based on Kabat, Chothia, and IMGT numbering schemes.

[0260] In one embodiment, the modifications introduced into the VH and CH1 and / or VL and CL domains are complementary to each other. The complementarity of the heavy and light chain interfaces can be based on spatial and hydrophobic contacts, electrostatic / charge interactions, or a combination of multiple interactions. Complementarity between protein surfaces is widely described in the literature as lock-and-bond coordination, knob-to-pore, protrusion-and-cavity, donor-and-acceptor, etc., all of which suggest a structural and chemical matching nature between two interacting surfaces.

[0261] In one embodiment, one or more introduced modifications introduce new hydrogen bonds at the interface across the Fab component. In one embodiment, one or more introduced modifications introduce new salt bridges at the interface across the Fab component. Exemplary alternatives are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0262] In some embodiments, the Fab domain includes a 192E substitution in the CH1 domain and 114A and 137K substitutions in the CL domain, the substitutions introducing a salt bridge between the CH1 and CL domains (see, for example, Golay et al., 2016, Journal of Immunology 196:3199-211).

[0263] In some embodiments, the Fab domain includes 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, the substitutions being used to exchange the hydrophobic and polar contact regions between the CH1 and CL domains (see, for example, Golay et al., 2016, Journal of Immunology 196:3199-211).

[0264] In some embodiments, the Fab domains may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces that promote proper assembly of the Fab domains (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, 39K and 62E modifications are introduced in the VH domain, H172A and F174G modifications are introduced in the CH1 domain, 1R, 38D, and (36F) modifications are introduced in the VL domain, and L135Y and S176W modifications are introduced in the CL domain. In another embodiment, 39Y modification is introduced in the VH domain and 38R modification is introduced in the VL domain.

[0265] The Fab domain can also be modified to replace the natural CH1:CL disulfide bonds with engineered disulfide bonds, thereby improving the pairing efficiency of Fab components. For example, engineered disulfide bonds can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, for example, Mazor et al., 2015, MAb 7:377-89).

[0266] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote proper assembly. For example, Wu et al., 2015, MAb 7:364-76, described replacing the CH1 domain with the constant domain of the T cell receptor and the CL domain with the b domain of the T cell receptor, and replacing these domains with additional charge-charge interaction pairs between the VL and VH domains by introducing 38D modification in the VL domain and 39K modification in the VH domain.

[0267] Instead of using a Fab heterodimerization strategy to facilitate proper VH-VL pairing, or in addition to using a common light chain (also referred to as a universal light chain), a VL of the common light chain can be used in each Fab VL region of the ABM of this disclosure. In various embodiments, employing a common light chain as described herein reduces the number of inappropriate types of ABM compared to employing a pristine homologous VL. In various embodiments, the VL domains of the ABM are identified from monospecific antibodies comprising the common light chain. In various embodiments, the VH region of the ABM comprises an in vivo rearranged human heavy chain variable gene segment in mouse B cells previously engineered to express a limited library of human light chains or a single human light chain homologous to the human heavy chain, and to generate an antibody library containing multiple human VHs homologous to one or both of two possible human VLs, wherein the antibody library is specific to the antigen of interest. The common light chain is a light chain derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, and contains a somatic mutant (e.g., affinity maturation) version. See, for example, U.S. Patent No. 10,412,940.

[0268] In some embodiments, the Fab is in the form of a single-chain Fab (“scFab”), which typically comprises VH, CH1, VL, CL, and a linker. In some embodiments, the domains of the scFab are arranged in the following N-terminal to C-terminal order: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. The linker can be as described in Section 6.2.3 and is preferably at least 30 amino acids, and in some respects, between 32 and 50 amino acids. The single-chain Fab domains are stabilized by native disulfide bonds between the CL and CH1 domains.

[0269] 6.2.2.scFv

[0270] Single-chain Fv or “scFv” antibody fragments include both the VH and VL domains of the antibody within a single polypeptide chain, enabling them to be expressed as single-chain polypeptides while retaining the specificity of the intact antibody from which they originate. Typically, scFv polypeptides further include a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for target binding. Examples of suitable linkers for connecting the VH and VL chains of scFV are the linkers identified in Section 6.2.3.

[0271] Unless otherwise stated, as used herein, scFv may have VL and VH variable regions in any order. For example, relative to the N-terminus and C-terminus of the peptide, scFv may include VL-connector-VH or may include VH-connector-VL.

[0272] scFv can include VH and VL sequences from any suitable species, such as mouse, human, or humanized VH and VL sequences.

[0273] To generate nucleic acids encoding scFv, DNA fragments encoding VH and VL are operatively linked to another fragment encoding a linker, for example, any linker described in Section 6.2.3 (typically a repeat of a sequence containing the amino acids glycine and serine, such as the amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 4), such that the VH and VL sequences can be expressed as a continuous single-stranded protein, wherein the VL and VH regions are linked by a flexible linker (see, for example, Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proceedings of the National Academy of Sciences 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0274] 6.2.3. Connector

[0275] In some respects, this disclosure provides ABMs, wherein two or more domains (e.g., Fab and Fc regions) are linked to each other via linker (or “spacer”) peptides. Such linkers are referred to herein as “ABM linkers”, in contrast to antibody-drug conjugate (“ADC”) linkers for linking drugs to ABMs, as described, for example, in Section 6.4.

[0276] Peptide linkers (e.g., polyglycine) are well known in the art and typically allow for the proper folding of one or both components of a fusion protein. Linkers provide flexible connecting regions for the fusion protein components, allowing the two ends of the molecule to move independently and can play an important role in maintaining the proper function of each part. Thus, in some cases, the connecting region acts both as a linker that combines the two parts together and as a spacer that allows each of the two parts to form its own biological structure without interfering with the other part.

[0277] The range of ABM linkers can be from 2 amino acids to 60 or more amino acids, and in some respects, the length of the peptide linker can range from 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids.

[0278] This disclosure provides an ABM comprising a first polypeptide and a second polypeptide (e.g., the first polypeptide and the second polypeptide of the embodiments described in Section 6.2), each polypeptide comprising a first linker and a second linker. The first linker and the second linker may have a length of 0 to 60 or 0 to 50 amino acids, such as 0, 1, 2, 3, 4, 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 The number of amino acids can be 1, 46, 47, 48, 49, or 50, for example, 0-10, 5-15, 10-20, 15-25, 0-30, 5-30, 10-30, 20-30, 0-40, 5-40, 10-40, 15-40, 20-40, 25-40, 30-40, 35-40, 0-50, 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, or 45-50. For ABMs in Fc-Fab, clamp, and tandem Fab forms, the typical linker length is between 5 and 30, for example, 5-30 amino acid residues. For the extended form of ABM, the typical linker length is 25 to 45, for example, 30-40 amino acid residues.

[0279] Charged (e.g., charged hydrophilic connectors) and / or flexible connectors are particularly preferred. Examples of flexible connectors that can be used in the ABM of this disclosure include those disclosed in the following literature: Chen et al., 2013, *Advanced Drug Delivery Review* 65(10):1357-1369 and Klein et al., 2014, *Protein Engineering, Design & Selection* 27(10):325-330. Particularly useful flexible connectors are repeats of glycine and serine, such as monomers or polymers of GnS or SGn, where n is an integer from 1 to 18, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. The most common GnS or SGn is (G4S). n (G4S is disclosed as SEQ ID NO: 3) (i.e. (Gly4Ser) n or (Gly–Gly–Gly–Gly–Ser) n ) joint, where n represents the number of repetitions of the motif.

[0280] Extended joints containing four, five, six or more repeats (e.g., six, seven, eight, nine or ten or more repeats) of G4S (disclosed as SEQ ID NO: 3) and / or another flexible joint motif are particularly useful for extended forms, wherein the extended joints act as spacers and are believed to provide more flexible binding, resulting in greater affinity and / or cohesion for small soluble molecules.

[0281] In some embodiments, the ABM connector is a polyglycine connector, such as Gly-Gly, Gly-Gly-Gly (3Gly), 4Gly (SEQ ID NO: 5), 5Gly (SEQ ID NO: 6), 6Gly (SEQ ID NO: 7), 7Gly (SEQ ID NO: 8), 8Gly (SEQ ID NO: 9) and 9Gly (SEQ ID NO: 10).

[0282] In other embodiments, the ABM linker is a glycine-serine linker. Examples of such linkers also include Ser-Gly, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO: 11), Ser-Gly-Gly-Gly (SEQ ID NO: 12), Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 3), Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 13), Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 14), Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 15), Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 16), Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 17). 17), (Gly-Gly-Gly-Gly-Ser) n (G4S is disclosed as SEQ ID NO:3) and (Ser-Gly-Gly-Gly-Gly)n (SG4 is disclosed as SEQ ID NO:13), where n (the number of motif repetitions) = 1 to 10. (Gly-Gly-Gly-Gly-Ser) n (G4S is disclosed as SEQ ID NO: 3) and (Ser-Gly-Gly-Gly-Gly) n (SG4 is disclosed as SEQ ID NO: 13) and is also referred to as (G4S). n and (SG4) n In one embodiment, the peptide linker is (Gly-Gly-Gly-Gly-Ser)1 (SEQ ID NO: 3), (Gly-Gly-Gly-Gly-Ser)2 (SEQ ID NO: 18), (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 4), or (Gly-Gly-Gly-Gly-Ser)4 (SEQ ID NO: 19). In some embodiments, the first linker and the second linker have the same amino acid sequence. In some embodiments, the polyglycine and serine amino acid sequence comprises 2 to 6 repeats of the GGGGS (SEQ ID NO: 3) amino acid sequence, such as 2, 3, 4, 5, or 6 repeats of the GGGGS (SEQ ID NO: 3) amino acid sequence. For the extended form, extended linkers containing 4, 5, 6, or more repeats (e.g., 6, 7, 8, 9, or 10 or more repeats) of any of the above motifs are considered.

[0283] 6.2.4. Hinge Area

[0284] In other embodiments, the ABM of this disclosure includes a hinge region, for example, a hinge region consisting of two hinged structural domains. The hinge can be used to connect the Fab structural domain to the Fc structural domain or to stabilize the ABM configuration.

[0285] The hinge region can be natural or modified. The hinge region is typically located at the N-end of the Fc region; however, in some embodiments, the hinge region may additionally or alternatively exist at the C-end of the Fc region of the ABM of this disclosure, for example in… Figure 13B and Figure 13C In the Fc-Fab configuration described in [the text].

[0286] The native hinge region is the hinge region typically found between the Fab and Fc domains of naturally occurring antibodies. A modified hinge region is any hinge region whose length and / or composition differs from that of the native hinge region. Such hinges can contain hinge regions derived from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat hinge regions. Other modified hinge regions can include complete hinge regions derived from antibodies of a different class or subclass than the heavy chain Fc region. Alternatively, a modified hinge region can include a portion of a native hinge or repeating unit, where each unit in the repeating unit is derived from the native hinge region. In another alternative, the native hinge region can be modified by converting one or more cysteine ​​or other residues to neutral residues, such as serine or alanine, or by converting appropriately placed residues to cysteine ​​residues. In this way, the number of cysteine ​​residues in the hinge region can be increased or decreased. Other modified hinge regions can be fully synthetic and can be engineered to have desired properties, such as length, cysteine ​​composition, and flexibility.

[0287] Many modified hinge areas have been described in, for example, U.S. Patent Nos. 5,677,425, WO 99 / 15549, WO2005 / 003170, WO 2005 / 003169, WO 2005 / 003170, WO 98 / 25971 and WO 2005 / 003171, which are incorporated herein by reference.

[0288] In various embodiments, positions 233-236 within the hinge structure domain can be G, G, G and empty; G, G, empty and empty; G, empty, empty and empty; or all empty, wherein the positions are numbered according to EU numbers.

[0289] In some embodiments, the ABM of this disclosure includes a modified hinge domain that has reduced binding affinity to the Fcγ receptor compared to a wild-type hinge domain of the same isotype (e.g., human IgG1 or human IgG4).

[0290] In one embodiment, the Fc region of one or both chains of the ABM disclosed herein has a complete hinge structure domain at its N end.

[0291] In one embodiment, the Fc region and hinge region of the ABM of this disclosure are both derived from lgG4, and the hinge region includes the modified sequence CPPC (SEQ ID NO: 20). Compared to lgG1 containing the sequence CPPC (SEQ ID NO: 20), the core hinge region of human lgG4 contains the sequence CPSC (SEQ ID NO: 21). Serine residues present in the lgG4 sequence increase the flexibility of this region, thus allowing a portion of the molecule to form disulfide bonds within the same protein chain (intra-chain disulfide bonds), rather than bridging with other heavy chains in the IgG molecule to form inter-chain disulfide bonds (Angel et al., 1993, *Molecular Immunology* 30(1):105-108). Replacing serine residues with proline to provide the same core sequence as lgG1 allows for complete inter-chain disulfide bond formation in the lgG4 hinge region, thereby reducing the heterogeneity of the purified product. This modified isotype is called lgG4P.

[0292] 6.2.5. Interlocking Hinge Sequence

[0293] The hinge area can be a mating hinge area.

[0294] For example, a chimeric hinge may include an “upper hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4, which is combined with a “lower hinge” sequence derived from the hinge region of human IgG1, human IgG2, or human IgG4.

[0295] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 22) (formerly disclosed as SEQ ID NO: 8 of WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 23) (formerly disclosed as SEQ ID NO: 9 of WO2014 / 121087). Such chimeric hinge sequences may suitably be linked to the IgG4 CH2 region (e.g., by incorporation into the IgG4 Fc domain, such as the human or mouse Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, as described in Section 6.2.7.1).

[0296] 6.2.6. Hinge sequences with reduced effector functionality

[0297] In another embodiment, the hinge region may be modified to reduce the effector function, for example, as described in WO2016161010A2, which is incorporated herein by reference in its entirety. In various embodiments, the modified hinge region positions 233-236 are G, G, G and empty; G, G, empty and empty; G, empty, empty and empty; or all empty, wherein the positions are numbered according to EU numbers (as shown in Figure 1 of WO 2016161010A2). These segments may be represented as GGG-, GG--, G---, or ----, where "-" indicates an empty position.

[0298] Position 236 is vacant in classical human IgG2, but is occupied in other classical human IgG isotypes. In all four human isotypes, positions 233-235 are occupied by residues other than G (as shown in Figure 1 of WO 2016161010A2).

[0299] The hinge modification at positions 233-236 can be combined with position 228, which is occupied by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but by S in human IgG4 and by R in human IgG3. The S228P mutation in IgG4 antibody is beneficial for stabilizing IgG4 antibody and reducing the exchange of heavy and light chain pairs between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0300] The exemplary hinge region has residues 226-236, sometimes referred to as the middle (or core) and lower hinge, which are occupied by modified hinge sequences of GGG-(233-236), GG--(233-236), G---(233-236) and no G(233-236). Optionally, the hinge domain amino acid sequence includes CPCPAPGGG-GPSVF (SEQ ID NO: 24) (previously disclosed as SEQ ID NO: 1 of WO 2016161010A2), CPCPAPGG--GPSVF (SEQ ID NO: 25) (previously disclosed as SEQ ID NO: 2 of WO 2016161010A2), CPCPAPG---GPSVF (SEQ ID NO: 26) (previously disclosed as SEQ ID NO: 3 of WO2016161010A2) or CPCPAP----GPSVF (SEQ ID NO: 27) (previously disclosed as SEQ ID NO: 4 of WO2016161010A2).

[0301] The modified hinge region described above can be incorporated into the heavy chain constant region, which typically contains CH2 and CH3 domains and may have additional hinge segments (e.g., upper hinges) located on either side of the designated region. Such additional constant region segments typically belong to the same isotype, preferably a human isotype, but can also be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but can also be human IgG1, IgG2, or IgG3, or hybrids thereof, where the domains belong to different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2-4 of WO2016161010A2.

[0302] In specific embodiments, the modified hinge sequence can be linked to the IgG4 CH2 region (e.g., by incorporation into the IgG4 Fc domain, such as the human or mouse Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, as described in Section 6.2.7.1).

[0303] 6.2.7. Fc structural domain

[0304] The ABM disclosed herein can include an Fc region derived from any suitable species. In one embodiment, the Fc region is derived from a human Fc domain.

[0305] The Fc domain can be derived from any suitable antibody class, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0306] The two Fc domains within the Fc region may be identical or different from each other. In natural antibodies, the Fc domains are usually identical, but in order to generate antigen-binding molecules, such as the ABM of this disclosure, the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.2.7.2 below.

[0307] In natural antibodies, the heavy chain Fc domain of IgA, IgD, and IgG consists of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc domain of IgE and IgM consists of three heavy chain constant domains (CH2, CH3, and CH4). These dimers form an Fc region.

[0308] In the ABM of this disclosure, the Fe region and / or the Fc domain therein may include heavy chain constant domains from one or more different classes of antibodies (e.g., one, two, or three different classes).

[0309] In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG1.

[0310] In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG2.

[0311] In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG3.

[0312] In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG4.

[0313] In one embodiment, the Fc region includes a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0314] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0315] It should be understood that the heavy chain constant domain of the Fc region used to generate the ABM of this disclosure may contain variants of the aforementioned naturally occurring constant domains. Such variants may include one or more amino acid variations compared to the wild-type constant domain. In one instance, the Fc region of this disclosure includes at least one constant domain that is sequence-different from the wild-type constant domain. It should be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another instance, the variant constant domain is at least 70% identical or similar. In another instance, the variant constant domain is at least 80% identical or similar. In another instance, the variant constant domain is at least 90% identical or similar. In yet another instance, the variant constant domain is at least 95% identical or similar.

[0316] IgM and IgA are naturally occurring covalent polymers of a common H2L2 antibody unit in humans. When IgM is incorporated into the J chain, it exists as a pentamer, or as a hexamer when the J chain is absent. IgA exists as both monomers and dimers. The heavy chains of IgM and IgA have 18 amino acids extending to a C-terminal constant domain, referred to as the tailpiece. The tailpiece contains cysteine ​​residues that form disulfide bonds between the heavy chains in the polymer and are considered to play an important role in polymerization. The tailpiece also contains a glycosylation site. In some embodiments, the ABM of this disclosure does not include the tailpiece.

[0317] The Fc domain incorporated into the ABM of this disclosure may include one or more modifications that alter protein functional properties, such as binding to Fc receptors like FcRn or leukocyte receptors, binding to complement, modified disulfide bond structures, or altered glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.2.7.1.

[0318] The Fc domains can also be modified to include modifications that improve the manufacturability of asymmetric ABMs, for example, by allowing heterodimerization, where dissimilar Fc domains are preferred over identical Fc domains. Heterodimerization allows the generation of ABMs in which different ABS are interconnected through Fc regions containing Fc domains with different sequences. Section 6.2.7.2 illustrates an example of a heterodimerization strategy.

[0319] It should be understood that any of the above modifications can be combined in any suitable manner to achieve the desired functional properties and / or combined with other modifications to change the properties of ABM.

[0320] 6.2.7.1. Fc domain with altered effector function

[0321] In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.

[0322] In a particular embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a particular embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically, human FcγRIIIa, FcγRI, or FcγR1a, most specifically, human FcγR11a. In one embodiment, the effector function is selected from one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and cytokine secretion. In a particular embodiment, the effector function is ADCC.

[0323] In one embodiment, the Fc region includes an amino acid substitution (numbered according to the Kabat EU index) at a location selected from the group consisting of E233, L234, L235, N297, P331, and P329. In a more specific embodiment, the Fc region includes an amino acid substitution (numbered according to the Kabat EU index) at a location selected from the group consisting of L234, L235, and P329. In some embodiments, the Fc region includes amino acid substitutions L234A and L235A (numbered according to the Kabat EU index). In one such embodiment, the Fc region is an Igd Fc region, specifically a human Igd Fc region. In one embodiment, the Fc region includes an amino acid substitution at location P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, specifically P329G (numbered according to the Kabat EU index). In one embodiment, the Fc region includes an amino acid substitution at position P329 and additional amino acid substitutions (numbered according to the Kabat EU index) at positions selected from E233, L234, L235, N297, and P331. In a more specific embodiment, the additional amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc region includes amino acid substitutions (numbered according to the Kabat EU index) at positions P329, L234, and L235. In a more specific embodiment, the Fc region includes amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”).

[0324] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Therefore, in a particular embodiment, each Fc domain of the Fc region includes amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbers), meaning that in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (numbered according to the Kabat EU index).

[0325] In one embodiment, the Fc domain is the IgG1 Fc domain, specifically the human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 that includes D265A, N297A mutations (EU number) to reduce effector function.

[0326] In another embodiment, the Fc domain is an IgG4 Fc domain with reduced binding to the Fc receptor. An exemplary IgG4 Fc domain with reduced binding to the Fc receptor may include an amino acid sequence selected from Table C below. In some embodiments, the Fc domain contains only the bolded portion of the sequence shown below:

[0327]

[0328]

[0329]

[0330] In a particular embodiment, the IgG4 with reduced effector function comprises the bold portion of the amino acid sequence of SEQ ID NO: 31 of WO2014 / 121087 (corresponding to amino acids 99-326 of SEQ ID NO: 31 of the present invention) and is sometimes referred to herein as IgG4s or hIgG4s.

[0331] For heterodimer ABM, combinations of the above-mentioned variant IgG4 Fc sequences can be incorporated, such as the Fc region of the combination of SEQ ID NO: 30 of WO2014 / 121087 (or its bold portion, corresponding to amino acids 99-329 of SEQ ID NO: 30 of this application) and SEQ ID NO: 37 of WO2014 / 121087 (or its bold portion, corresponding to amino acids 99-329 of SEQ ID NO: 32 of this application), or the Fc region of the combination of SEQ ID NO: 31 of WO2014 / 121087 (or its bold portion, corresponding to amino acids 99-326 of SEQ ID NO: 31 of this application) and SEQ ID NO: 38 of WO2014 / 121087 (or its bold portion, corresponding to amino acids 99-326 of SEQ ID NO: 33 of this application).

[0332] 6.2.7.2. Fc heterodimer variant

[0333] Many multispecific molecular forms require dimerization between two Fc domains, which, unlike native immunoglobulins, are operatively linked to different antigen-binding domains (or portions thereof, e.g., VH or VH-CH1 of Fab). Insufficient heterodimerization of the two Fc regions can be a barrier to increasing the yield of desired multispecific molecules and represents a challenge for purification. Various methods available in the art can be used to enhance the dimerization of the Fc domains that may be present in the ABM of this disclosure, for example, as disclosed in EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO2009 / 089004A1.

[0334] This disclosure provides an ABM comprising Fc heterodimers, i.e., Fc regions comprising heterologous, dissimilar Fc domains. Heterodimerization strategies are used to enhance dimerization of Fc regions (or portions thereof, e.g., VH or VH-CH1 of Fab) operatively linked to different ABS, and to reduce dimerization of Fc domains operatively linked to the same ABS. Typically, each Fc domain in the Fc heterodimer comprises the CH3 domain of the antibody. The CH3 domain is derived from a constant region of any isotype, class, or subclass of antibody, preferably of the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in previous sections.

[0335] Heterodimerization of two different heavy chains at the CH3 domain produces the desired ABM, while homodimerization of the same heavy chain reduces the yield of the desired ABM. Therefore, in a preferred embodiment, the two haptens that associate to form the ABM of this disclosure will contain a modified CH3 domain, which, relative to the unmodified chain, promotes heterodimer association.

[0336] In specific embodiments, the modification promoting Fe heterodimer formation is a so-called "knob-in-hole" or "knob-in-hole" modification, comprising a "knob" modification in one Fc domain and a "hole" modification in another Fc domain. Knob-in-hole techniques are described in, for example, U.S. Patent No. 5,731,168; US 7,695,936; Ridgway et al., 1996, *Protein Engineering* 9:617-621; and Carter, 2001, *Immunol Meth* 248:7-15. Typically, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity to promote heterodimer formation and inhibit homodimer formation. A protrusion is constructed by replacing a small amino acid side chain at the interface of the first peptide with a larger side chain (e.g., tyrosine or tryptophan). A compensating cavity of the same or similar size as the protrusion is created at the interface of the second peptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0337] Therefore, in some embodiments, amino acid residues in the CH3 domain of the first subunit of the Fc domain are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit, which can be positioned in a cavity within the CH3 domain of the second subunit, and amino acid residues in the CH3 domain of the second subunit of the Fc domain are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit, in which the protrusion in the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residues having a larger side chain volume are selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues having a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusions and cavities can be formed by altering the nucleic acid encoding the polypeptide (e.g., through site-specific mutagenesis or through peptide synthesis). An exemplary substitution is Y470T.

[0338] In a specific embodiment of this type, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). Optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbered according to the Kabat EU index). In another embodiment, in the first Fc domain, the serine residue at position 354 is further replaced with a cysteine ​​residue (S354C), or the glutamate residue at position 356 is replaced with a cysteine ​​residue (E356C) (specifically, the serine residue at position 354 is replaced with a cysteine ​​residue), and in the second Fc domain, the tyrosine residue at position 349 is further replaced with a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). In a particular embodiment, the first Fc domain includes amino acid substitutions S354C and T366W, and the second Fc domain includes amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU index).

[0339] In some embodiments, electrostatic orientation (e.g., as described in Gunasekaran et al., 2010, J Biool Chem 285 (25): 19637-46) can be used to promote the association of the first and second subunits of the Fc domain.

[0340] Alternatively, or in addition to using a modified Fc domain to promote heterodimerization, the Fc domain can be modified to allow for selective purification strategies for Fc heterodimers. In one such embodiment, a half-antibody includes a modified Fc domain that eliminates its binding to protein A, thereby enabling a purification method that produces a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. Thus, the ABM includes a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces the binding of the ABM to protein A compared to a corresponding ABM lacking this amino acid difference. In one embodiment, the first CH3 domain binds to protein A, and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as H95R modification (via IMGT exon number; H435R, via EU number). The second CH3 may further include Y96F modification (via IMGT; Y436F, via EU). Thus, this type of modification is referred to herein as a “star” mutation.

[0341] 6.3. Target Molecules

[0342] The ABM of this disclosure includes at least two Fab domains, Fab1 and Fab2, each of which specifically binds to a target molecule, such as a small soluble molecule. In some embodiments, the ABM of this disclosure further includes two additional Fab domains, Fab3 and Fab4, which may be bound or unbound. In some embodiments, the target molecule bound by Fab1, Fab2 (and, when present, the bound forms of Fab3 and Fab4) is a protein molecule.

[0343] Preferably, Fab1 and Fab2 are selected such that they can each specifically bind to their respective epitopes simultaneously. In some embodiments, Fab1 and Fab2 specifically bind to different target molecules, such as a pair of molecules that can interact with each other (e.g., tumor-associated antigen and CD3). In other embodiments, Fab1 and Fab2 bind to the same target molecule, different epitopes, or the same epitope.

[0344] It is believed that the ABM of this disclosure is particularly advantageous for binding small molecular weight proteins, such as proteins with a molecular weight less than 100 kDa, less than 75 kDa, or less than 60 kDa (with or without post-translational modifications such as glycosylation). In specific embodiments, the proteins bound by the ABM of this disclosure have molecular weights ranging from 5 kDa to 75 kDa, 5 kDa to 60 kDa, 5 kDa to 45 kDa, 5 kDa to 30 kDa, 10 kDa to 75 kDa, 10 kDa to 60 kDa, 10 kDa to 45 kDa, or 10 kDa to 30 kDa, and in each case may or may not contain post-translational modifications such as glycosylation.

[0345] Exemplary target molecules that Fab1 and / or Fab2 can bind to include ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRLI, ADORA2A, Aggrecan, AGR2, AICDA, AIF1, AIG1, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, AZGP1 (zinc-α-glycoprotein), ART-4, B7, B7.1, B7.2, BAD, BAFF, BAGI, BAIi, BCL2, BCL6, BDNF, BLNK, BLR1 (… MDRlS), BlyS, BMPl, BMP2, BMP3B (GDF10), BMP4, BMP6, BMPS, BMPR1A, BMPR1B, BMPR2, BPAG1 (lectin), BRCA1, Ba-733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-I, CASP-8 / m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CDS, CD8, CDI-IA, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, C D154, CDC27, CDK-4 / m, CDKN2A, CXCR4, CXCR7, CXCL12, C19orf10 (IL27w), C3, C4A, CS, CSR1, CANT1, CASPI, CASP4, CAV1, CCBP2 (D6 / JAB61), CCLI (I-309 CCLII (chemokine), CCL13 (MCP-4), CCLIS (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCLIS (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MIP-2), SLC, exodus-2, CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / chemokine-2), CCL2S (TECK), CCL26 (chemokine-3), CCL27 (CTACK / ILC), CCL2S,CCL3 (MIP1a), CCL4 (MIP-1b), CCLS (RANTES), CCL7 (MCP-3), CCLS (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1 / HM14S), CCR2 (mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCRS (CMKBRSI ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EB1), CCRS (CMKBRS / TER1 / CKR-LI), CCR9 (GPR-9-6), CCRLI (VS HK1), CCRL2 (L-CCR), CD164, CDlC, CD200, CD-22, CD24, CD2S, CD3S, CD3E, CD3G, CD3Z, CD4, CD44, CD4SRB, CD47, CD4S, CDS2, CD 69. CD72, CD79A, CD79B, CDSO, CDS1, CDS3, CDS6, CD137, CD13S, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CDH1 (E-cadherin White), CDH10, CDH12, CDH13, CDH1S, CDH19, CDH20, CDHS, CDH7, CDHS, CDH9, CDK2, CDK3, CDK4, CDKS, CDK6, CDK7, CDK9, CDKN1A (p21 Wap1 / Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEBPB, CER1, CHGA, CHGB, chitinase, CHST1O, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSFS, CKLFSF6, CKLFSF7, CKLFSFS, CLDN3, CLDN7 (blockin-7), CLN3, CLU (clusterin), CMKLR1, CMKOR1 (R DC1), CNR1, COLISA1, COLIA1, COL4A3, COL6A1, CR2, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA-4, CTNNB1 (β-catenin), CTSB (cathepsin B), CX3CLI (SCYD1), CX3CR1 (V2S), CXCLI (GRO1), CXCLIO (IP-10), CXCL11 (I-TAC / IP-9), CXCL13, CXCL14, CXCL16CXCL2 (GR02), CXCL3 (GR03), CXCLS (ENA-7S / LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), CYBS, CYC1, CYSLTR1, HIF-1-a, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-met, DAB2IP, DES, DKFZp4S1J011S, DNCLI, DPP4, DAM, EGFR, EGFRvll l, EGP-1, EGP-2, ELF2-M, Ep-CAM, E2F1, ECGF1, EDG1, EFNA1, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, EN01, EN02, EN03, EPHB4, EPO, EREG, ERKS, E SR1, ESR2, F3 (TF), FADD, FasL, FASN, FCER1A, FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FG F1S, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGFS, FGF7 (KGF), FGFS, FGF9, FGFR3, FIGF (VEGFD), FILI (EPSILON), FILI (ZETA), FLJ12SS4, FLJ2SS30, FLRT1 (fibronectin), FOS, FOSLI (FRA-1), FY (DARC), Flt-I, Flt-3, folate receptor, G250 antigen, GAGE, GROB, GABRP (GAB Aa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GDFS, GFil, GGT1, GM-CSF, GNAS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPRS1 (FKSGSO), GRCC10 (C10), GRP, GSN (gelatin), GSTP1, HAVCR2, HDAC4, HDACS, HDAC7A, HDAC9, HGF, HIP1 histamine and histamine receptor, HLA-A, HLA-DRA, HM74, HMOX1, HUMCYT2A, HLA-DR, HMI 24, human chorionic gonadotropin (HCG) and its subunits, HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M,HST-2 or 1a, IGF-IR, IFN-ɣ, IFN-α, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IGBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-1, IL-10, IL-10RA, IL-10RB, IL-11, IL-11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL-13RA1, IL-13RA2, IL-14, IL-1S, IL-1SRA, IL-16, IL-17, IL-17B, IL-17C, IL-17R, IL-18, IL-18BP, IL-18R1, IL-18RAP, IL-19, IL-IA, IL-1B, IL-1F10, IL-1FS, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-1HY1, IL-1R1, IL-1R2, IL-1RAP, IL-1RAPL1, IL-1RAPL2, IL-1RL1, IL-1RL2 IL-1RN, IL-2, IL-20, IL-20RA, IL-21R, IL-22, IL-22R, IL-22RA2, IL-23, IL-24, IL-2S, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-2RA, IL-2RB, IL-2RG, IL-3, IL-30, IL-3RA, IL-4, IL-4R, IL-S, IL-5RA, IL-6, IL-6R, IL-6ST (glycoprotein 130), IL-7, IL-7R, IL-S, IL-SRA, IL-SRB, IL-9, IL-9R, IL-K, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (a6 antigen), ITGAV, ITGB3, ITGB4 (b4 integrin), insulin-like growth factor-I (IGF-1), ICEBERG, ICOS L, ID2, IFN-a, IFNA1, IFNA2, IFNA4IFNAS, IFNA6, IFNA7, IFNB1, IFNW1, JAG1, JAK1, JAK3, JUN, K6HF, KAi1, KDR, KITLG, KLFS (GC box BP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK1S, KLK3, KLK4, KLKS, KLK6, KLK9,KRT1, KRT19 (keratin 19), KRT2A, KRTHB6 (hair-specific type II keratin), KC4 antigen, KS-1 antigen, KS 1-4, Le-Y, LDR / FUT, LAMAS, LEP (leptin), Lingo-p7S, Lingo-Troy, LPS, LTA (TNF-β), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​MACMARCKS, MAG or Omgp, MAP2K7 (c-Jun), MDK, MIB1, metaphase factor, MIF, MIP-2, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionein-III), MTSS1, MUC1 (mucin), MYC, MYD88, macrophage migration Migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ES0-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1 / 2, MU M-3, NCA66, NCA95, NCA90, NCK2, Neuron, NFKB1, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgR-Nogo66 (Noga), NgRp7S, NgR-Troy, NME1 (NM23A), NOXS , NPPB, NROB1, NROB2, NR1D1, NR1D2, NRIH2, NRIH3, NRIH4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4 A1, NR4A2, NR4A3, NRSA1, NRSA2, NR6A1, NRP1, NRP2, NTSE, NTN4, ODZ1, OPRD1, PCSK9, P2RX7, PAP, PART1, PATE, PAWR, PCA3, PCNA, PD-1, PD-L1, α4β7, OX40, GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDFS, CGRP, Lingo-I, Factor IXa, Factor X, ICOS, GARP, BTLA, CD160, RORI, 2B4, KIR, CD27, OX40, A2aR, PDGFA, PDGFB, PECAM1, PF4 (CXCL4), PGF, PGR, phosphatidylcholine, PIAS2, PIK3CG, PLAU (uPA), PLG, PLXDC1, PPBP (CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL, PROCPROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2 (COX-2), PTN, pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, 10 PIGF, ILGF, ILGF-IR, IL-6, RS5, RANTES, RAC2 (p21Rac2), RARB, RGS1, RGS13, RGS3, RNF110 (ZNF144), ROB02, S100A2, SCGB1D2 (lipophilic B), SCGB2A1 (mammary globin 2), SCGB2A2 (mammary globin 1), SCYE1 (endothelial monocyte activating cytokine), SDF2, SERPINA1, SERPINA3, SERPINABS (mammary filament inhibitory protein), SERPINE1 (PAI-1), SERP INF1, SHBG, SLA2, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPP1, SPRR1B (Sprl), ST6GAL1, STAB1, STATE, STEAP, STEAP2, TIOI, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tendinogen, TRAIL receptor, TNF-α, Tn-antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, TB4R2, TBX21, TCP10, TDGF1, TEK, TGFA, TGFB1 TGFBlil, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, THBS1 (thromboretin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor, TLR10, TLR2, TLR3, TLR4, TLRS, TLR6, TLR7, TLRS, TLR9, TNF, TNF-α, TNFAIP2 (B94), TNFAIP3, TNFRSF11A, TNFRSF1A, TNFRSF1B, TNFRSF21, TN FRSFS, TNFRSF6 (Fas), TNFRSF7, TNFRSFS, TNFRSF9, TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF1S (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSFS (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSFS (CD30 ligand)TNFSF9 (4-lBB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TPS3, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAPS, TRAF6, TREM1, TREM2, TRPC6, TSLP, TWEAK, VEGFR, ED-B fibronectin, WT-1, 17-IA antigen, complement factors C3, C3a, C3b, C5a, CS, angiogenesis markers, bcl-2, bcl-6 Kras, cMET, CD19 / CD3, BCMA / CD3, EGFR, HER3, IL17RA / IL7R, IL-6 / IL-23, IL1 / IL-8, IL-6, IL-6R / IL-21, IL-21R, ANG2 / VEGF, VEGF / PDGFR-β, vascular endothelial growth factor (VEGF) receptor 2 / CD3, PSMA / CD3, EPCAM / CD3, VEGFR-1, VEGFR-2, VEGFR-3, VEGFB, VEGFC, multifunctional proteoglycans, VHL CS, VLA-4, c-FMS / CSFIR, RET, HER3, HER4, IGFR, PDGFR, c-KIT, BCR, integrins, MMPs, VEGF, EGF, PIGF, PDGF, HGF, angiopoietin, ERBB-3 / C-MET, ERBB-2 / C-MET, EGF receptor 1 / CD3, EGFR / HER3, PSCA / CD3, C-MET / CD3, ENDOSIALIN / CD3, EPCAM / CD3, IGF-1R / CD3, FAPALPHA / CD3, EGFR / IGF-IR, IL 25 17A / F, EGF receptor 1 / CD3 and CD19 / CD16, KHI, Tn-antigen, TF-antigen, CD44, glycolipids, glycosphingolipids, such as 30 Gg3, Gb3, GD3, GD2, Gb5, Gm1, Gm2, sialytetrasaccharide ceramide, XCL1 (lymphocyte chemokine), XCL2 (SCM-1b), XCR1 (GPRS / CCXCR1), YY1, and ZFPM2.

[0346] In some embodiments, the ABM of this disclosure is capable of binding a pair of target molecules, for example, via Fab1 and Fab2. Exemplary target molecule pairs include CD137 and CD20, CD137 and EGFR, CD137 and Her-2, CD137 and PD-1, CD137 and PDL-1, VEGF and PD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1, TIM-3 and PDL-1, EGFR and DLL-4, CD138 and CD20, CDI 38 and CD40, CDI 9 and CD20, CD20 and CD3, CD3 and CD33, CD3 and CD133, CD47 and CD20, CD38 and CD138, CD38 and CD20, CD20 and CD22, CD38 and CD40, CD40 and CD20, CD-8 and IL-6, CSPGs and RGM. A. CTLA-4 and BTN02, IGF1 and IGF2, IGF1 / 2 and Erb2B, IGF-1R and EGFR, EGFR and CD13, IGF-1R and ErbB3, EGFR-2 and IGFR, VEGFR-2 and Met, VEGF-A and angiopoietin-2 (Ang-2), IL-12 and TWEAK, IL-13 and IL-1β, PDGFR and VEGF, EpCAM and CD3, Her2 and CD3, CD19 and CD3, EGFR and Her3, CD16a and CD30, CD30 and PSMA, EGFR and CD3, CEA and CD3, TROP-2 and HSG, TROP-2 and CD3, MAG and RGM A, NgR and RGM A, NogoA and RGM A, OMGp and RGM A. PDL-1 and CTLA-4, CTLA-4 and PD-1, PD-1 and TIM-3, RGMA and RGM B. Te38 and TNFa, TNFa and Blys, TNFa and CD-22, TNFa and CTLA-4 domains, TNFa and GP130, TNFa and IL-12p40, and TNFa and RANK ligand.

[0347] In some embodiments, the ABM of this disclosure can bind to one or more cytokines, cytokine-related proteins, and / or cytokine receptors, such as one or a pair of cytokines, cytokine-related proteins, and / or cytokine receptors, for example, via Fab1 and Fab2. Exemplary cytokines, cytokine-related proteins, and / or cytokine receptors include BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, F... GF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFNG, IFNW1, FILI, FILI (EPSILON), FILI (ZET A), ILIA, ILIB, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, ILIO, ILi1, ILI2A, ILI2B, ILI3, ILI4, ILI5, ILI6, ILI7, ILI7B, ILI8, ILI9, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, FGER1, FGFR2, FGFR3, EGFR, RORI, 2B4, KIR, CD137, CD27, OX40, CD4 0L, A2aR, CD48, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CD70, CD40, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (T NF-α), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD), VEGF, VEGFB,VEGFC、ILIR1、ILIR2、ILIRLI、ILIRL2、IL2RA、IL2RB、IL2RG、IL3RA、IL4R、IL5RA、IL6R、IL 7R, IL8RA, IL8RB, IL9R, ILIORA, ILIORB, IL11RA, ILI2RB1, ILI2RB2, ILI3RA1, ILI3RA2, ILI5RA, ILI7R, ILI8R1, IL20RA, IL2 1R, IL22R, IL1HY1, ILIRAP, ILIRAPLI, ILIRAPPL2, ILIRN, IL6S T, ILI8BP, ILI8RAP, IL22RA2, AIF1, HGF, LEP (protein), PTN, THPO.

[0348] In some embodiments, the ABM of this disclosure can bind to one or more chemokines, chemokine-related proteins, and / or chemokine receptors, such as one or a pair of chemokines, chemokine-related proteins, and / or chemokine receptors, for example, via Fab1 and Fab2. Exemplary chemokines, chemokine-related proteins, and chemokine receptors include CCLI (I-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCLII (chemokine), CCLI3 (MCP-4), CCLI5 (MIP-1d), CCLI6 (HCC-4), CCLI7 (TARC), and CCLI 8 (PARC), CCLI9 (MIP-3b), CCL20 (MIP-3a), CCL21 (SLC / exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / chemokine-2), CCL25 (TECK), CCL26 (chemokine-3), CCL27 (CTACK / ILC), CCL28, CXCLI (GRO1), CXCL2 (GR02), CXCL3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCLIO (IP)10), CXCL11 (I-TAC), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1 (lymphocyte chemokine), XCL2 (SCM-1b), BLR1 (MDR15), CCBP2 (D6 / JAB61), CCR1 (CKR1 / HM145), CCR2 ( mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6 ), CCR7 (CKR7 / EBI1), CCRS (CMKBR8 / TER1 / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR 1 (GPR5 / CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSGSO), C XCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), HM74, ILSRA (IL8Ra), ILSRB (IL8Rb), LTB4R (GPR1 6), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSFS, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HIF1A, ILS, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.

[0349] In some embodiments, the ABM of this disclosure is capable of binding a pair of cytokines, cytokine receptors, and / or cytokine-related proteins. Exemplary cytokine pairs include IL-1α and IL-1β, IL-12 and IL-18, TNFα and IL-23, TNFα and IL-13, TNF and IL-18, TNF and IL-12, TNF and IL-1β, TNF and MIF, TNF and IL-6, TNF and IL-6 receptor, TNF and IL-17, IL-17 and IL-20, IL-17 and IL-23, TNF and IL-15, TNF and VEGF, and V. EGFR and EGFR, PDGFR and VEGF, IL-13 and IL-9, IL-13 and IL-4, IL-13 and IL-5, IL-13 and IL-25, IL-13 and TARC, IL-13 and MDC, IL-13 and MIF, IL-13 and TGF-β, IL-13 and LHR agonists, IL-13 and CL25, IL-13 and SPRR2a, IL-13 and SPRR2b, IL-13 and ADAM8, as well as TNFα and PGE4, IL-13 and PED2, as well as TNF and PEG2.

[0350] In some embodiments, the ABM of this disclosure is capable of binding to at least two epitopes on a single cytokine, a cytokine receptor, or a cytokine-related protein. Exemplary cytokines include TSLP, IL-1a, IL-1β, IL-12, IL-18, TNFα, IL-23, IL-13, MIF, IL-6, IL-6 receptor, IL-17, IL-20, IL-15, VEGF, VEGFR, EGFR, PDGFR, IL-9, IL-4, IL-5, IL-25, TARC, MDC, TGF-β, LHR agonist, CL25, SPRR2a, SPRR2b, ADAM8, PGE4, PED2, and PEG2.

[0351] In some embodiments, the ABM of this disclosure is capable of binding to its antigenic target with an affinity similar to or greater than that of conventional forms of antibodies or antibody fragments.

[0352] In some embodiments, the ABM of this disclosure has agonist function against its target molecules. In other embodiments, the ABM of this disclosure has blocking and / or antagonistic function against its antigens or target molecules.

[0353] In some respects, relative to parental antibodies (or parental antibody pairs), such as relative to parental IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies from which the Fa of ABM originates, the ABM of this disclosure has an IC50 similar to or lower than that of its antigen or target molecule. 50For example, it has a similar or lower IC50 compared to the conventional IgG form. 50 .

[0354] In some embodiments, the ABM of this disclosure is bispecific for a single ligand and forms a 1:1 ligand complex at a higher level relative to one or more parental antibodies.

[0355] When Fab1 and Fab2 bind to different epitopes on the same target molecule, the binding to the target molecule is preferably non-competitive, i.e., Fab1 and Fab2 do not compete for binding to the target molecule (e.g., this may occur if the epitopes overlap). Assays for measuring binding competition between antibodies and antibody fragments are known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA), fluorescence activated cell sorting (FACS) assays, and surface plasmon resonance assays.

[0356] For example, real-time, label-free biolayer interferometry on the Octet HTX biosensor platform (Pall ForteBio) can be used to determine competition for binding to target molecules. In a specific embodiment of this assay, the entire assay was performed at 25°C in a buffer solution (HBS-EBT buffer) containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 1 mg / mL BSA, 0.05% v / v surfactant Tween-20, pH 7.4, with the plate shaken at 1000 rpm. To assess whether two antibodies or their antigen-binding fragments can compete to bind to corresponding epitopes on their specific target antigens, the pent-His-labeled target antigen (“pent-His” disclosed as SEQ ID NO: 34) was first captured into an Octet biosensor tip (Fortebio, #18-5122) coated with an anti-pent-His antibody (“pent-His” disclosed as SEQ ID NO: 34) by immersing the biosensor tip in a well containing a first antibody or its antigen-binding fragment (hereinafter referred to as Ab-1). The captured antigen was then saturated with Ab-1 by immersion in a well containing a solution (e.g., 50 µg / mL solution) of a first antibody or its antigen-binding fragment (hereinafter referred to as Ab-2). The biosensor tip was then immersed in a well containing a second antibody or its antigen-binding fragment (hereinafter referred to as Ab-2) (e.g., 50 μg / mL solution). The biosensor tip was washed in HBS-EBT buffer between each step of the assay. The binding reaction can be monitored in real time throughout the assay, and the binding reaction at the end of each step can be recorded. The binding reaction of Ab-2 with the target antigen pre-complexed with Ab-1 can be compared, and the competitive / non-competitive behavior of different antibody / antigen binding fragments against the same target antigen can be determined.

[0357] In various embodiments:

[0358] ●ABM (e.g., type A ABM or type B ABM) does not contain Fab3 and Fab4, and Fab1 and Fab2 bind to the same or different epitopes on the same target molecule;

[0359] ●ABM (e.g., type A ABM or type B ABM) does not contain Fab3 and Fab4, and Fab1 and Fab2 bind to different target molecules;

[0360] ●ABM (e.g., C-type ABM) contains unbound Fab3 and unbound Fab4, and Fab1 and Fab2 bind to the same or different epitopes on the same target molecule.

[0361] ●ABM (e.g., C-type ABM) contains unbound Fab3 and unbound Fab4, and Fab1 and Fab2 bind to different target molecules;

[0362] ●ABM (e.g., C-type ABM) contains bound Fab3 and bound Fab4, and Fab1 and Fab2 bind the same epitope, while Fab3 and Fab4 bind the same epitope but different from the epitope bound by Fab1 and Fab2, whether on the same target molecule as the target molecule bound by Fab1 and Fab2 or on a different target molecule.

[0363] ●ABM (e.g., C-type ABM) contains bound Fab3 and bound Fab4, with Fab1 and Fab3 binding the same epitope, while Fab2 and Fab4 bind the same epitope but different from the epitope bound by Fab1 and Fab3, whether on the same target molecule as the target molecule bound by Fab1 and Fab3 or on a different target molecule.

[0364] When two or more of Fab1, Fab2, Fab3, and Fab4 bind to the same epitope on a target molecule, such Fab domains may have the same or different heavy chain CDR sequences and / or the same or different VH sequences. Optionally, they may have the same or different VL sequences.

[0365] Unbound by theory, it is believed that the ABM of this disclosure has the advantage of binding target molecules with greater affinity compared to parental monospecific or bispecific antibodies with natural conformations. Therefore, in some embodiments, the ABM of this disclosure can bind one or more target molecules with greater affinity compared to parental monospecific or bispecific antibodies with natural conformations. For example, in some embodiments, the ABM can have a lower Kb of binding to the target molecule in cell-based binding assays compared to the corresponding parental monospecific or bispecific antibody. D And / or have a stronger EC50 value (e.g., as described in Section 7).

[0366] The agonist or antagonist activity of a given antibody or ABM depends on target selection, epitope coverage, and form selection. For example, the identification of agonist and antagonist antibodies can be achieved through function-based screening. The ABM forms disclosed herein are particularly advantageous for antagonist activity against small soluble molecules.

[0367] 6.4. Antibody-drug conjugates

[0368] The ABM disclosed herein can be conjugated to a drug moiety, for example, via a connector, particularly where the ABM is intended to be used as a cancer therapeutic agent. For convenience, such conjugates are referred to herein as antibody-drug conjugates (or “ADCs”).

[0369] In some respects, the drug portion exerts cytotoxic or cell-inhibiting activity. In one embodiment, the drug portion is selected from maytansinoid, kinin-like protein KIF11 inhibitors, V-ATPase (vacuole H+-ATPase) inhibitors, pro-apoptotic agents, Bcl2 (B-cell lymphoma 2) inhibitors, MCL1 (myeloid leukemia 1) inhibitors, HSP90 (heat shock protein 90) inhibitors, IAP (inhibitor of apoptosis) inhibitors, mTOR (rapamycin mechanotarget) inhibitors, microtubule stabilizers, microtubule destabilizers, olistatin, dolalastatin, MetAP (methionine aminopeptidase), CRM1 Chromosome maintenance 1 inhibitors, DPPIV (dipeptidyl peptidase IV) inhibitors, proteasome inhibitors, inhibitors of mitochondrial phosphotransferase, protein synthesis inhibitors, kinase inhibitors, CDK2 (cyclin-dependent kinase 2) inhibitors, CDK9 (cyclin-dependent kinase 9) inhibitors, kinin inhibitors, HDAC (histone deacetylase) inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove adhesives, RNA polymerase inhibitors, topoisomerase inhibitors, or DHFR (dihydrofolate reductase) inhibitors.

[0370] In some embodiments, the cytotoxic agent is maytansine having the following structure:

[0371] .

[0372] In some embodiments, the cytotoxic agent is maytansine having the following structure:

[0373] .

[0374] In some embodiments, the ADC includes ABM and...

[0375]

[0376] in It is a key to ABM.

[0377] In some embodiments, the antibody-drug conjugate includes ABM of this disclosure, and

[0378]

[0379] in It is a key to ABM.

[0380] In some embodiments, the ADC includes ABM and...

[0381] or

[0382] or

[0383] Its mixture,

[0384] in It is a key to ABM disclosed herein.

[0385] In some embodiments, the bond is linked to ABM via the sulfur component of the cysteine ​​residue.

[0386] In some embodiments, the bond is linked to ABM via the nitrogen component of the lysine residue.

[0387] In the ADC of this disclosure, cytotoxic agents and / or cell inhibitors are linked to ABM via ADC adapters. The ADC adapters of the ABM linking the cytotoxic agents and / or cell inhibitors to the ADC can be short, long, hydrophobic, hydrophilic, flexible, or rigid, or can be composed of segments each independently possessing one or more of the aforementioned properties, such that the adapters can contain segments with different properties. The adapters can be multivalent, such that they covalently link more than one agent to a single site on the ABM, or monovalent, such that they covalently link a single agent to a single site on the ABM.

[0388] In some respects, the connector is selected from cuttable connectors, non-cuttable connectors, hydrophilic connectors, front-energized connectors, or dicarboxylic acid-based connectors.

[0389] As those skilled in the art will understand, the ADC connector links the cytotoxic agent and / or cell inhibitor to ABM by forming a covalent bond with the cytotoxic agent and / or cell inhibitor at one site and a covalent bond with ABM at another site. The covalent bond is formed through a reaction between functional groups on the ADC connector and functional groups on the agent and ABM.

[0390] ADC adapters are preferably, but not necessarily, chemically stable to extracellular conditions and can be designed to specifically cleave, sacrifice, and / or otherwise degrade within cells. Alternatively, ADC adapters not designed for specific intracellular cleavage or degradation can be used. The choice between stable and unstable ADC adapters may depend on the toxicity of the cytotoxic agent and / or cell inhibitor. For agents toxic to normal cells, stable adapters are preferred. For selective or targeted agents with low toxicity to normal cells, the chemical stability of the ADC adapter to the extracellular environment is less important. In the case of ADCs, a variety of ADC adapters that can be used to bind drugs to ABM are known in the art. Any of these ADC adapters, as well as other ADC adapters, can be used to bind cytotoxic agents and / or cell inhibitors to the ABM of the ADCs disclosed herein.

[0391] Exemplary multivalent ADC linkers that can be used to link numerous cytotoxic agents and / or cell inhibitors to a single ABM molecule are described in the following references: e.g., WO 2009 / 073445; WO 2010 / 068795; WO 2010 / 138719; WO2011 / 120053; WO 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379; WO2014 / 093394; WO 2014 / 093640, the contents of which are incorporated herein by reference in their entirety. For example, the Fleximer linker technology developed by Mersana et al. has the potential to achieve high-DAR ADCs with favorable physicochemical properties. The Mersana technology is based on incorporating drug molecules into a solubilized polyacetal backbone via a series of ester bonds. This method can exhibit high-load ADC (DAR up to 20) while maintaining good physicochemical properties.

[0392] Exemplary monovalent ADC adapters that can be used are described in the following literature: for example, Nolting, 2013, “Antibody-Drug Conjugates”, Methods in Molecular Biology 1045:71-100; Ducry et al., 2010, Bioconjugate Chem. 21:5-13; Zhao et al., 2011, J. Med. Chem. 54:3606-3623; U.S. Patent No. 7,223,837; U.S. Patent No. 8,568,728; U.S. Patent No. 8,535,678; and WO2004010957, each of which is incorporated herein by reference.

[0393] As examples and not limitations, the following describes some cuttable and non-cuttable ADC connectors that may be included in the ADCs disclosed herein.

[0394] In some embodiments, the selected ADC adapter is cleavable in vivo. Cleavable ADC adapters may contain chemically or enzymatically unstable or degradable bonds. Cleavable ADC adapters typically rely on intracellular processes to release the drug, such as cytoplasmic reduction, exposure to acidic conditions in lysosomes, or cleavage by specific intracellular proteases or other enzymes. Cleavable ADC adapters typically incorporate one or more chemical bonds that can be chemically or enzymatically cleaved, while the remainder of the ADC adapter is incleavable. In some embodiments, the ADC adapter includes chemically unstable groups, such as hydrazones and / or disulfides. Adapters including chemically unstable groups utilize the differential properties between plasma and certain cytoplasmic compartments. Intracellular conditions that promote drug release from hydrazone-containing ADC adapters are acidic environments in endosomes and lysosomes, while disulfide-containing ADC adapters are reduced in cytosols containing high concentrations of thiols (e.g., glutathione). In some embodiments, the plasma stability of ADC adapters including chemically unstable groups can be increased by introducing steric hindrance using substituents near the chemically unstable groups.

[0395] A cleavable ADC connector may contain uncleavable portions or segments, and / or cleavable portions or segments may be incorporated into other uncleavable ADC connectors to make them cleavable. For example, polyethylene glycol (PEG) and related polymers may contain cleavable groups in the polymer backbone. For instance, a polyethylene glycol or polymer ADC connector may contain one or more cleavable groups, such as disulfides, hydrazones, or dipeptides.

[0396] Other degradable bonds that may be included in the ADC linker include ester bonds formed by the reaction of PEG carboxylic acid or activated PEG carboxylic acid with an alcohol group on a bioactive agent, wherein such ester groups are typically hydrolyzed under physiological conditions to release the bioactive agent. Hydrolyzable degradable bonds include, but are not limited to, carbonate bonds; imine bonds resulting from the reaction of amines and aldehydes; phosphate ester bonds formed by the reaction of alcohols with phosphate groups; acetal bonds as products of the reaction of aldehydes and alcohols; orthoester bonds as products of the reaction of formate esters and alcohols; and oligonucleotide bonds formed by a phosphoramidite group (including, but not limited to, at the polymer terminus) and the 5' hydroxyl group of an oligonucleotide.

[0397] In some embodiments, the ADC adapter includes an enzyme-cleavable peptide moiety, such as a tripeptide or dipeptide. In specific embodiments, the dipeptide is selected from: Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Val-Lys; Ala-Lys; Phe-Cit; Leu-Cit; lle-Cit; Phe-Arg; and Trp-Cit. In some embodiments, the dipeptide is selected from: Cit-Val; and Ala-Val.

[0398] In any of the various embodiments of the ADC discussed above or herein, the drug:antibody ratio of the ADC (or, in this example, the drug:ABM ratio) can be from 1 to 20, more typically in the range of 2 to 10.

[0399] 6.5. Nucleic Acids and Host Cells

[0400] In another aspect, this disclosure provides nucleic acids encoding the ABM of this disclosure. In some embodiments, the ABM is encoded by a single nucleic acid. In other embodiments, the ABM is encoded by multiple nucleic acids (e.g., two, three, four or more).

[0401] A single nucleic acid can encode an ABM comprising a single polypeptide chain, an ABM comprising two or more polypeptide chains, or a portion of an ABM comprising more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an ABM comprising three, four, or more polypeptide chains, or three polypeptide chains of an ABM comprising four or more polypeptide chains). For individual control of expression, open reading frames encoding two or more polypeptide chains can be under the control of individual transcriptional regulatory elements (e.g., promoters and / or enhancers). Open reading frames encoding two or more polypeptides can also be controlled by the same transcriptional regulatory elements, separated by internal ribosome entry sites (IRES) sequences, thereby allowing translation into individual polypeptides.

[0402] In some embodiments, an ABM comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the ABM may be equal to or less than the number of polypeptide chains in the ABM (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0403] The nucleic acid disclosed herein may be DNA or RNA (e.g., mRNA).

[0404] On the other hand, this disclosure provides host cells and vectors containing the nucleic acids of this disclosure. The nucleic acids may be present in the same host cell or in a separate host cell, or in a single vector or a separate vector, as described in more detail below.

[0405] 6.5.1. Carrier

[0406] This disclosure provides vectors comprising nucleotide sequences encoding the ABM or ABM components described herein (e.g., one or both of the polypeptide chains of a half-antibody). Vectors may include, but are not limited to, viruses, plasmids, viscera, λ phages, or yeast artificial chromosomes (YACs).

[0407] A variety of vector systems can be used. For example, one type of vector utilizes DNA elements derived from animal viruses, such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous Sarcoma Virus, MMTV, or MOMLV), or SV40 virus. Another type of vector utilizes RNA elements derived from RNA viruses, such as Semliki Forest virus, Eastern Equine Encephalitis virus, and flaviviruses.

[0408] Furthermore, cells that have stably integrated their DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. For example, markers can provide auxotrophic hosts with resistance to auxotrophs, biocidal agents (e.g., antibiotics), or resistance to heavy metals such as copper. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Optimal mRNA synthesis may require additional elements. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0409] Once an expression vector or DNA sequence containing the construct has been prepared for expression, the expression vector can be transfected or introduced into suitable host cells. This can be achieved using various techniques, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. The methods and conditions used to culture the resulting transfected cells and recover the expressed peptide are known to those skilled in the art and can be varied or optimized according to the specific expression vector and mammalian host cells used, as described in this specification.

[0410] 6.5.2. Cells

[0411] This disclosure also provides host cells that include the nucleic acids disclosed herein.

[0412] In one embodiment, the host cell is genetically engineered to include one or more nucleic acids as described herein.

[0413] In one embodiment, the host cell is genetically engineered using an expression cassette. The phrase "expression cassette" refers to such a sequence that can influence gene expression in a host compatible with its nucleotide sequence. Such a cassette may contain a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in influencing expression, such as, for example, inducible promoters, may also be used.

[0414] This disclosure also provides host cells including the vectors described herein.

[0415] The cells can be, but are not limited to, eukaryotic cells, bacterial cells, insect cells, or human cells. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0416] 6.6. Pharmaceutical Compositions

[0417] The ABM and / or ADC disclosed herein may be in the form of a composition comprising ABM and / or ADC and one or more carriers, excipients, and / or diluents, optionally used with one or more other pharmaceutical agents that provide improved transfer, delivery, tolerability, etc. The composition may be formulated for a specific purpose, such as for human pharmaceutical use or veterinary use. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the ABM and / or ADC, and for therapeutic use, on the mode of administration.

[0418] The dosage of antigen-binding molecules (such as monospecific or bispecific antigen-binding molecules) administered to a patient can vary depending on the patient's age and body size, target disease, symptoms, route of administration, etc. Preferred dosages are typically calculated based on body weight or body surface area. When the bispecific antigen-binding molecule of this disclosure is used for therapeutic purposes in adult patients, it may be advantageous to administer the antigen-binding molecule of this disclosure intravenously in a single dose of about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight. The frequency and duration of treatment can be adjusted according to the severity of the symptoms. Effective dosages and schedules for administering antigen-binding molecules can be determined empirically; for example, patient progression can be monitored through periodic assessments, and the dosage adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0419] For therapeutic use, the composition may be provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the desired method of administration to the patient). The pharmaceutical composition may be administered to the patient via a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, local, or regional. In any given case, the most suitable route of administration will depend on the specific antibody and / or ADC, the subject, the nature and severity of the disease, and the subject's physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0420] The pharmaceutical composition can be conveniently available in unit dosage forms containing a predetermined amount of the ABM and / or ADC of this disclosure. The amount of ABM and / or ADC contained in the unit dose will depend on the disease being treated and other factors well known in the art. Such unit doses can be in the form of a lyophilized powder containing a predetermined amount of ABM and / or ADC suitable for a single administration, or in liquid form. The powder unit dosage form can be packaged in a kit with a syringe, a suitable diluent, and / or other components suitable for administration. The liquid unit dose can be conveniently provided in the form of a syringe pre-filled with a predetermined amount of ABM and / or ADC suitable for a single administration.

[0421] The pharmaceutical composition can also be supplied in bulk from an amount of ADC suitable for multiple administrations.

[0422] Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing ABM and / or ADC of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as “carriers”), i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and other miscellaneous additives commonly used in the art. See Remington's Pharmaceutical Sciences, 16th edition (edited by Osol, 1980). Such additives should be non-toxic to the recipient at the dosage and concentration employed.

[0423] Buffers help maintain pH within a range close to physiological conditions. They can be present in a variety of concentrations, but are typically present in concentrations ranging from about 2 mM to about 50 mM. Buffers suitable for use in this disclosure include organic and inorganic acids and their salts, such as citrate buffers (e.g., mixtures of monosodium citrate and disodium citrate, mixtures of citrate and trisodium citrate, mixtures of citrate and monosodium citrate, etc.), succinate buffers (e.g., mixtures of succinic acid and monosodium succinate, mixtures of succinic acid and sodium hydroxide, mixtures of succinic acid and disodium succinate, etc.), tartrate buffers (e.g., mixtures of tartaric acid and sodium tartrate, mixtures of tartaric acid and potassium tartrate, mixtures of tartaric acid and sodium hydroxide, etc.), and fumarate buffers (e.g., mixtures of fumaric acid and monosodium fumarate, etc.). Fumarate-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc., gluconate buffers (e.g., gluconate-sodium gluconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.), oxalate buffers (e.g., oxalate-sodium oxalate mixtures, oxalate-sodium hydroxide mixtures, oxalate-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can be used.

[0424] Preservatives may be added to slow microbial growth, and said preservatives may be added in an amount of about 0.2%-1% (w / v). Preservatives suitable for use in this disclosure include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium chloride (e.g., chloride, bromide, and iodide), hexamethyl ammonium chloride, and alkyl esters of parabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes referred to as “stabilizers,” may be added to ensure the isotonicity of the liquid compositions of this disclosure, and said isotonic agents comprise polyols, such as ternary or higher sugar alcohols, such as glycerol, erythritol, araitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients, functionally ranging from fillers to dissolving agents or additives that help prevent denaturation or adhesion to container walls. Typical stabilizers can be polyols (listed above); amino acids (such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.); organic sugars or sugar alcohols (such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc.); including cyclic alcohols, such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents; Examples of stabilizers include urea, glutathione, alpha-lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight peptides (e.g., peptides with 10 or fewer residues); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, sucrose, and trehalose; and trisaccharides such as raffinose; as well as polysaccharides such as dextran. Stabilizers may be present in amounts from 0.5 to 10 wt% per weight of ADC.

[0425] Nonionic surfactants or detergents (also known as "wetting agents") can be added to help dissolve glycoproteins and protect them from agitation-induced aggregation. This also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxoisomerics (184, 188, etc.), and pluronic polyols. Nonionic surfactants can be present in concentrations ranging from about 0.05 mg / mL to about 1.0 mg / mL, for example, from about 0.07 mg / mL to about 0.2 mg / mL.

[0426] Other miscellaneous excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and solubilizers.

[0427] 6.7. Indications for Treatment

[0428] The ABM, ADC, and pharmaceutical compositions disclosed herein can be used to treat conditions associated with antigens or target molecules that are bound by the ABM of this disclosure, such as conditions associated with abnormal expression or activity of the antigen or target molecule or abnormal cells or tissues expressing said antigen or target molecule. The ABM, ADC, and pharmaceutical compositions of this disclosure can be administered to subjects in need, such as humans or non-human animals exhibiting symptoms or markers of one or more conditions associated with abnormal expression or activity of antigens or target molecules that are bound by ABM.

[0429] In some embodiments, the ABM, ADC, or pharmaceutical composition of this disclosure is administered to treat any disease or condition in which stimulation, activation, and / or targeting of an antigen or target molecule is required. In specific embodiments, the ABM of the present invention can be used to treat, prevent, and / or improve any disease or condition associated with or mediated by the expression or activity of an antigen or target molecule.

[0430] The ABM disclosed herein can be illustrated by an ABM containing one of multiple Fab domains (e.g., Fab1 and / or Fab2) that bind thymic stromal lymphopoietin (“TSLP”) (e.g., human TSLP). TSLP is an immune cytokine that induces CD4-mediated growth in dendritic cells with a pre-allogeneic phenotype. + T cell response (Gilliet et al., 2003, *Journal of Experimental Medicine* 197(8):1059-1063). TSLP is involved in the initiation of allergic inflammation (Watanabe et al., 2004, *Nature Immunology* 5:426-434). TSLP acts on multiple cell types (e.g., dendritic cells, CD4+). + T cells, eosinophils, basophils, mast cells, and type 2 innate lymphocytes (ILC2) (Mjosberg et al., 2012, *Immunity* 37(4):649-59) drive inflammation, specifically type 2 inflammation (characterized by the production of cytokines IL-5, IL-13, and IL-4). Type 2 inflammation is characteristic of asthma and other allergic diseases such as atopic dermatitis and Netherton syndrome. TSLP has been found to induce fibroblast accumulation and collagen deposition in animals, demonstrating its additional role in promoting fibrotic conditions.

[0431] Therefore, ABM as a TSLP antagonist can be used to treat inflammation, specifically allergic inflammation, and fibrotic conditions. ABM that binds to TSLP (monospecific or bispecific) can be used to treat TSLP-related symptoms in subjects of need (e.g., human subjects). Exemplary conditions associated with TSLP signaling include asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, Netherton syndrome, eosinophilic esophagitis (EoE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, cancer, rheumatoid arthritis, COPD, systemic sclerosis, keloids, ulcerative colitis, chronic sinusitis (CRS), nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, Churg-Strauss syndrome, eosinophilic myalgia syndrome, eosinophilic syndrome, eosinophilic granuloma with polyangiitis, and inflammatory bowel disease.

[0432] Example

[0433] 7.1. Example 1: Construction of Alternative Antigen-Binding Molecules

[0434] Non-competitive parental mAbs were selected to construct alternative bispecific ABMs for human TSLP, a protein belonging to the cytokine family with a molecular weight of approximately 15-18 kDa (depending on glycosylation state). These parental mAbs shared a common light chain. All heterodimeric bispecific ABMs were constructed with a "button-in-pore" mutation in the Fc region to promote Fc heterodimer formation (Merchant et al., 1998, *Nature Biotechnology* 16:677-681). For the Fc-Fab form ( Figure 1B and Figure 2B ), by using (G4S) n The connector links the VH-CH1 fragment to the C-terminus of the Fc to construct a heavy chain (G4S is disclosed as SEQ ID NO: 3), n=1-6. In clamp form ( Figure 3B In this process, the internal Fab segment is inert and does not bind to the TSLP. This inert Fab segment is replaced with a flexible long (G4S) in an extended form. n Connector (G4S is disclosed as SEQ ID NO: 3), n=6-8 ( Figure 3A In the 2+2 tandem Fab form, all four Fab fragments are functional and can bind to the antigen. Figures 3C-3D The short connectors between Fab segments in clamp and 2+2 tandem Fab configurations are 2xG4S (SEQ ID NO: 18) or 3xG4S (SEQ ID NO: 4).

[0435] Similarly, non-competitive parent mAbs were selected for constructing bispecific Fc-Fabs of human ligand X. These parent anti-ligand X mAbs share a common light chain.

[0436] For cell surface targets, Fc-Fab is constructed in a similar manner using a Fab fragment of a common light chain that shares bispecific Fc-Fab and a constant region of hIgG4 (shown as hIgG4s) (US9359437B2) or hIgG1 with reduced effector functions.

[0437] All antibodies using the human IgG4 constant region contain S228P (EU) substitution in the hinge region to minimize hemi-antibody formation (Labrijn et al., 2009, Nature Biotechnology 27:767-771).

[0438] 7.2. Example 2: Expression of antigen-binding molecules

[0439] All alternative forms of bispecific ABM were expressed in Expi293F cells (Thermo Fisher Scientific) via transient transfection. ABM in the Expi293F supernatant was purified using a ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) equipped with a HiTrap rProteinA FF column (GE Healthcare). After single-step elution, the ABM was neutralized, dialyzed to a final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted, and stored at -80°C.

[0440] 7.3. Example 3: Activity of anti-hTSLP bispecific ABM in bioassays

[0441] The ability of purified anti-hTSLP bispecific ABM to inhibit hTSLP activity in a luciferase reporter assay was evaluated. Baf3 cells stably expressing hIL-7R, hTSLP, and STAT3-luciferase reporter genes were seeded at 40,000 cells / well in IL-3-free medium and incubated overnight. For hTSLP dose-response mapping, serially diluted 1:3 hTSLP was added to each well, starting at a final hTSLP concentration of 10 nM. Figure 4ATo determine the blocking activity of anti-hTSLP ABM, a “Race-form” blocking assay was used, in which ABM and hTSLP were simultaneously added to reporter cells. Anti-hTSLP ABM was serially diluted 1:3, with each antibody starting at a final concentration of 100 nM. Human TSLP was added to a constant concentration approximately at the EC50 of the TSLP dose-response curve. After incubation for 5.5 hours, the plate was equilibrated at room temperature for 15 minutes. 100 μl of One-Glo substrate (Promega) was added to each well. After incubation at room temperature for 5 minutes, luminescence was measured on Envision. The activity of the three non-competitive anti-hTSLP parental antibodies 30206, 30217, and 30230 was shown in the STAT3-luciferase reporter assay. Figure 4B Three bispecific pairings using these parental mAbs were tested. Conventional hIgG4 bispecific antibodies showed similar blocking activity to the corresponding parental antibody combinations. Figure 5 For each bispecific pair, all alternative forms of bispecific ABM showed better blocking activity than conventional hIgG4 bispecific antibodies. Figures 6A-6C In general, for all bispecific pairings, the best form is Fc-Fab (with...). Figure 13A (as shown in the hinge configuration) and 2+2 tandem Fab-heterodimers. These ABMs have ICs... 50 The values ​​are summarized in Table 5-1.

[0442]

[0443] 7.4. Example 4: Size analysis of the in vitro complex formed between the anti-hTSLP bispecific antibody and recombinant hTSLP by asymmetric flow field coupled to multi-angle light scattering (A4F-MALLS) and flow fractionation.

[0444] 7.4.1. Overview

[0445] Size analysis of in vitro complexes formed between the following anti-TSLP ABMs and recombinant hTSLP (REGN4009) identified in Table 5-2 was performed using asymmetric flow field-flow fractionation coupled to multi-angle light scattering (A4F-MALLS): anti-TSLP parental Ab 30206 hIgG4, anti-TSLP parental Ab 30217 hIgG4, anti-TSLP parental Ab 30230 hIgG4, Fc-Fab_30206x30217-2xG4S (“2xG4S” is disclosed as SEQ ID NO: 18), Fc-Fab_30217x30230-2xG4S (“2xG4S” is disclosed as SEQ ID NO: 18). 18) Clamp type _30206x30217, clamp type _30217x30230, 2+2 tandem Fab_het(heterogeneous)_30206x30217 and 2+2 tandem Fab_het(heterogeneous)_30217x30230. In this study, the Fc-Fab ABM has Figure 13A The hinge type shown.

[0446] 7.4.2. Materials and Methods

[0447] 7.4.2.1. Molecules

[0448] Table 5-2 below lists the molecules analyzed by A4F-MALLS and their alternative names.

[0449]

[0450] 7.4.2.2. A4F-MALLS mobile phase buffer

[0451] A mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1) was prepared by mixing 1.4 g sodium dihydrogen phosphate monohydrate, 10.7 g disodium hydrogen phosphate heptahydrate, and 500 mL of 5 M sodium chloride. The solution was then brought to a final volume of 5.0 L with HPLC-grade water. The final measured pH of the buffer was 7.0. The mobile phase buffer was filtered (0.2 μm) before use.

[0452] 7.4.2.3.AF-MALLS

[0453] The A4F-MALLS system comprises an Eclipse™ 3+ A4F separation system coupled to an Agilent 1200 Series HPLC system equipped with a UV diode array detector, a Wyatt Technology Dawn HELEOS® II laser scattering (LS) detector, and an Optilab® T-rEX differential refractometer (RI) detector. The detectors are connected in series in the following order: UV-LS-RI. The LS and RI detectors are calibrated according to the instructions provided by Wyatt Technology.

[0454] Determined amounts of anti-TSLP mAb were individually combined with REGN4009 (recombinant TSLP) and diluted in 1X DPBS (pH 7.4) to produce an equimolar ratio of 1 μM anti-TSLP mAb : 1 μM hTSLP. Equimolar combinations of each parental mAb were prepared as stock solutions, and each stock solution was then mixed with an equimolar amount of hTSLP to produce a final solution concentration of 0.5 μM mAb1 + 0.5 μM mAb2 + 1 μM hTSLP. All samples were incubated at ambient temperature for 2 hours and kept unfiltered at 4°C before being injected into Eclipse™ short channels equipped with W350 spacer foil (350 μm spacer thickness, 2.2 cm spacer width) and using a 10 kDa MWCO regenerated cellulose membrane. Prior to injection of each sample, the channels were pre-equilibrated using mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1). Bovine serum albumin (BSA; 2 mg / mL; 10 μg loading) was injected separately and included as a system suitability control.

[0455] The fractionation method consists of four steps: injection, focusing, elution, and channel “rinsing”. A4F-MALLS mobile phase buffer (10 mM sodium phosphate, 500 mM sodium chloride, pH 7.0 ± 0.1) is used throughout the fractionation process. Each sample (7 μg) is injected at a flow rate of 0.2 mL / min for 1 min, followed by focusing at a focusing flow rate of 1.0 mL / min for 3 min. The sample is eluted at a channel flow rate of 1.0 mL / min for 15 min with a constant cross-flow of 3.0 mL / min, followed by elution over 5 min with a linear gradient cross-flow from 3.0 mL / min to 0 mL / min. Finally, the cross-flow is maintained at 0 mL / min for an additional 5 min to rinse the channels. BSA is fractionated using the same parameter settings.

[0456] 7.4.2.4. MALLS Data Analysis

[0457] Data were analyzed using ASTRA V software (version 5.3.4.14, Wyatt Technology). The data were fitted to an equation relating excess scattered light to solute concentration and weight-average molar mass Mw (Kendrick et al., 2001, *Anal Biochem.*, 299(2):136-46; Wyatt, 1993, *Anal.Chim.Acta*, 272(1):1-40):

[0458] Equation 1:

[0459] Where c is the solute concentration, R(θ,c) is the Raleigh ratio of the solute excess as a function of scattering angle and concentration, Mw is the molar mass, P(θ) describes the angle dependence of the scattered light (~1 for particles with a radius of gyration < 50 nm), A2 is the second virial coefficient of osmotic pressure expansion (negligible because the measurement is performed on a dilute solution), and...

[0460] Equation 2:

[0461] Where n0 represents the solvent refractive index, N A λ0 is Avogadro's number, λ0 is the wavelength of incident light in vacuum, and dn / dc represents a specific refractive index increment of the solute.

[0462] The molar mass of the BSA monomer was used to evaluate the calibration constants of the light scattering and differential refractive index detectors during data collection (system suitability check). The relative standard deviation (%RSD) of the average molar mass of the BSA determined from the UV and RI detectors is ≤5.0%.

[0463] The normalization coefficients, inter-detector delay volume, and band broadening terms for the light scattering detector were calculated from BSA chromatograms collected under the employed A4F-MALLS conditions. These values ​​were applied to data files collected for all other samples to correct for these terms.

[0464] The dn / dc values ​​and extinction coefficients at 215 nm or 280 nm (corrected for glycosylation) were experimentally determined using the protein conjugate analysis provided in Astra software. The corrected extinction coefficients and dn / dc values ​​were used to analyze all protein-protein complex samples.

[0465] 7.4.3. Results

[0466] A4F-MALLS was used to evaluate the relative size distribution of complexes formed between several anti-hTSLP ABMs and hTSLP. The theoretical molar mass and predicted stoichiometry of potential ABM complexes with hTSLP are shown in Tables 5-3 and 5-4:

[0467]

[0468]

[0469] As expected, when combined in equimolar ratios, each individual parental anti-TSLP mAb (H4H30206P2, H4H30217P2, H4H30230P2) forms typical 1:1 and 1:2 complexes with hTSLP (peak 3, ~179 kDa). Figure 7 (Table 5-5).

[0470]

[0471] However, when different combinations of the two parental mAbs (H4H30206P2 + H4H30217P2 and H4H30217P2 + H4H30230P2) were mixed with an equimolar amount of hTSLP, a heterogeneous distribution of the heterologous complex was observed. This indicates that each parental mAb can bind to the same molecule of hTSLP to form an extended antibody-antigen lattice in a process known as “paper doll re-dressing”. Figure 8 In these samples, a unique peak (peak 4) with a molar mass of approximately 342 kDa was observed, followed by a series of broad, poorly resolved species (peak 5) with molar mass distributions ranging from ~650 to 5000 kDa. Based on the calculated molar masses of each component, peak 4 can represent a 2:2 mAb:hTLSP complex, while peak 5 corresponds to a heterogeneous distribution of higher-order heterocomplexes composed of ≥4 mAb molecules coordinated with ≥3 hTSLP molecules (Tables 5-6).

[0472]

[0473] Furthermore, the complexes formed between hTSLP and a novel group of bispecific antibodies (TS-FC1-eL1, TS-FC6-eL2) were examined. These bispecific antibodies possess two distinct Fab domains derived from the same parental mAb combination tested above, linked to the C-terminus of the human Fc domain (Fc-Fab). Unlike the results obtained using the parental mAb combination, each Fc-Fab bispecific antibody (bsAb) primarily formed discrete 1:1 complexes with hTSLP (peak 3, ~178 kDa). Figure 9(Tables 5-7), while little or no additional higher-order complexes were observed (“paper doll dress-up”).

[0474]

[0475] This indicates that the two Fab domains on each Fc-Fab bsAb prefer to bind to the same molecule of hTSLP, thus forming a monocoordinated divalent interaction and thus ruling out the "paper doll re-dressing" process.

[0476] The complexation of two other sets of ABM forms disclosed herein with hTSLP was also evaluated in a similar manner. These two sets of ABM forms have an additional external Fab domain on each binding arm (2+2 tandem Fabs; TS-CL2-eL2, TS-CL3-eL2) or an additional internal non-binding Fab on each arm (clamp-type; TS-CL4-eL1, TS-CL6-eL1). Typically, each clamp-type ABM appears to form a 1:1 complex with hTSLP to some extent (peak 3, ~272 kDa). Figure 10 (Table 5-8); however, a wide range of heterogeneously distributed higher-order complexes (peak 5, ~650-7000 kDa) can also be detected in these samples. Figure 10 (Table 5-8) shows different degrees of "paper doll dress-up".

[0477]

[0478] Finally, when mixed with an equimolar amount of hTLSP, each 2+2 tandem Fab bsAb showed the highest “paper doll remake” tendency among all the novel bispecific forms tested. In these samples, a distinctive peak consistent with the free 2+2 tandem Fab bsAb (peak 2; ~255 kDa) was observed, followed by a series of broad, poorly resolved peaks representing a heterogeneous distribution of increasingly larger species, which terminated in very large complexes with molar masses exceeding ten (10) mega-daltons (peaks 4-5; ~500-14,000 kDa). Figure 11 (Table 5-9).

[0479]

[0480] 7.5. Example 5: Optimization of Connectors in hTSLP Fc-Fab

[0481] A series of bispecific Fc-Fabs against -hTSLP 30217x30230 were constructed using different adapters, ranging from a 2-amino acid GS adapter to a 30-amino acid 6xG4S adapter (SEQ ID NO: 38). The activity of these Fc-Fabs was evaluated in an hTSLP STAT3-luciferase reporter assay. Figure 12 Using a linker length between 2 and 5 x G4S (G4S is disclosed as SEQ ID NO: 3), optimal blocking activity of Fc-Fab was observed. Different hinge forms were also evaluated using a 30217x30230 bispecific Fc-Fab as an example (Figure 13). In form #1, the hinge sequence is located at the N-terminus of Fc-Fab, as it appears in the native hIgG4 sequence, with an S228P (EU) substitution ( Figure 13A In form #2, the same hinge sequence is removed from the N-terminus of the Fc-Fab and inserted into the C-terminus of the Fc CH3 domain and (G4S). n Between the connectors (G4S is disclosed as SEQ ID NO: 3) Figure 13B In form #3, the hinge is also located in the CH3 structural domain and (G4S). n Between the joints (G4S is disclosed as SEQ ID NO: 3). However, the upper hinge sequence is replaced with the G4S sequence (G4S is disclosed as SEQ ID NO: 3) Figure 13C All three hinge forms were combined with either a 1xG4S adapter (SEQ ID NO: 3) or a 4xG4S adapter (SEQ ID NO: 19) to construct bispecific Fc-Fabs against hTSLP 30217x30230. The activity of these Fc-Fabs was evaluated in an hTSLP STAT3-luciferase reporter assay. Figure 13D For Fc-Fab with 4xG4S connectors (SEQ ID NO: 19), different hinge configurations had little effect on their TSLP blocking activity, with hinge configuration #1 showing the best activity. For Fc-Fab with 1xG4S connectors (SEQ ID NO: 3), the IC50 of hinge configuration #1 was 5 to 10 times better than the other two hinge configurations.

[0482] 7.6. Example 6: Biacore analysis of Fc-Fab binding to Fc receptor

[0483] The equilibrium dissociation constants (K0) of different anti-TSLP Fc-Fab antibodies binding to purified recombinant human FcγR and FcRn receptor subtypes were determined using a MASS-2 (Bruker) / Biacore 3000 (GE Healthcare) biosensor based on real-time surface plasmon resonance.D The Fc-Fab constructs tested were TSLP 30206x30217 Fc_Fab 2xG4S (“2xG4S” is disclosed as SEQ ID NO: 18) (also known as REGN8759) and TSLP 30230x30217Fc_Fab 2xG4S (“2xG4S” is disclosed as SEQ ID NO: 18) (also known as REGN7860), as well as anti-FelD1(-)-IgG1 and IgG4 isotype controls (referred to as REGN1932 and REGN1945, respectively). The Fc receptors measured were human FcγRIIA(H131)-myc.6xHis, human FcγRIIA(R167)-10xHis, human FcγRIIB-myc.6xHis, human FcγRIIIA(F176)-myc.6xHis, human FcγRIIIB-mmh, human FcRn-mmh, and human FcγRI-6xHis.

[0484] 7.6.1. Materials and Methods

[0485] All binding studies were performed at 25°C in run buffers containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20 at pH 7.4 (HBS-ET) or PBS, 0.05% v / v surfactant Tween-20 at pH 6.0 (PBS-T-pH6.0). The surface of the MASS-2 / Biacore 3000 CM5 sensor was derivatized by amine conjugation with mouse anti-pentahistidine monoclonal antibody (“pentahistidine” disclosed as SEQ ID NO: 34) (GE Healthcare) or anti-myc monoclonal antibody (REGN642) to capture the extracellular domains of FcγR and FcRn receptors expressed using the C-terminal myc-myc-hexahistidine (“hexahistidine” disclosed as SEQ ID NO: 35) or histidine regions. Binding studies were conducted on different anti-TSLP Fc-Fab and wild-type Fc isotype controls. Different concentrations of anti-TSLP Fc-Fab (ranging from 5 µM to 0.3125 µM, 2-fold dilution), prepared in HBS-ET or PBS-T pH 7.4 and pH 6.0 running buffers, were injected at a flow rate of 50 μL / min onto the surfaces captured by FcγR and FcRn receptors. Binding of all anti-TSLP Fc-Fab to each of the captured FcγR and FcRn receptors was monitored for 1.5–2 min, and their dissociation in HBST running buffer was monitored for 10 min. At the end of each cycle, the surfaces captured by FcγR and FcRn receptors were regenerated by injection of 10 mM glycine-HCl pH 1.5 for 20–30 s, using mouse anti-pentahistidine monoclonal antibody (“pentahistidine” is disclosed as SEQ ID NO: 34) or anti-myc monoclonal antibody. All binding kinetics experiments were performed at 25 °C.

[0486] 7.6.2. Data Analysis

[0487] The dissociation equilibrium constant (KD) and dissociation half-life (t½) are calculated based on the kinetic rate as follows:

[0488] KD (M) = kd / ka and t½ (min) = ln2 / (60xkd)

[0489] 7.6.3. Results

[0490] The binding kinetics of different anti-TSLP Fc-Fab and control antibodies to different FcγR and FcRn receptors of this disclosure at 25°C are shown in Tables 5-10 and 5-11, respectively. In Table 5-10, NT indicates not tested and IC indicates indeterminate. In Table 5-11, NB indicates no binding and IC indicates indeterminate.

[0491]

[0492]

[0493] 7.7. Example 7: Pharmacokinetic evaluation of anti-TSLP Fc-Fab bispecific antibody in wild-type mice

[0494] 7.7.1. Overview

[0495] The pharmacokinetic profiles of two anti-TSLPFc-Fab bispecific binding molecules were evaluated in C57BL / 6 wild-type (WT) mice compared with the anti-fel d 1 IgG4 isotype control REGN1945, the unrelated conventional bispecific IgG4 control H4H21237D, and the hFcγ homodimer REGN1627: (1) 30206 x 30217 IgG4 Fc-Fab bispecific with a 2xG4S linker (SEQ ID NO: 18), also known as REGN8759; and (2) 30230 x 30217 IgG4 Fc-Fab bispecific with a 2xG4S linker (SEQ ID NO: 18), also known as REGN 8760.

[0496] 7.7.2. Materials and Methods

[0497] Five mice were included in each antibody cohort for each test. Mice administered REGN8759, REGN8760, REGN1945, and H4H21237D received a single subcutaneous (SC) dose of 1 mg / kg. Mice administered the hFcγ homodimer REGN1627 received a normalized SC dose based on molar equivalence (0.35 mg / kg) to the other antibodies in the study. Blood samples were collected at 6 hours post-administration and at days 1, 2, 3, 4, 7, 10, 14, and 21. Blood was processed into serum and frozen at -80°C until analysis. Total and functional serum concentrations of REGN8759 and REGN8760, as well as total serum concentrations of REGN1945, H4H21237D, and REGN1627, were measured using the GyroLab xPlore platform (Gyros).

[0498] Gyros technology utilizes affinity flow for automated immunoassays using laser-induced fluorescence detection. Samples are loaded onto a compression disc (CD) containing multiple radially arranged nano-level affinity trap columns. Liquid flow is controlled by centrifugal and capillary forces.

[0499] For measurements of total functional REGN8759 and REGN8760, and for measurements of total REGN1945, H4H21237D, and REGN1627 in serum, 100 μg / mL of test or control-specific biotinylated capture reagent (Table 5-11) was added to a Gyrolab Bioaffy 200 CD (Dynospheres) containing an affinity column pre-loaded with streptavidin-coated beads. Standards used for calibration (Table 5-11) were run in concentrations ranging from 0.488 to 2000 ng / mL. Serial dilutions of serum samples were prepared in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). Serial dilutions of standards were prepared in PBS + 0.5% BSA containing 2% normal mouse serum (NMS). At room temperature, a 1:50 dilution of serum sample singlets and copies of standards were added to an affinity column coated with the capture reagent. Captured human IgG was detected using Alexa-647-conjugated mouse anti-human IgG1 / hIgG4 monoclonal antibody (REGN2567, 0.5 μg / mL) diluted in Rexxip F buffer (Gyros); the resulting fluorescence signal was recorded in reaction units (RU) using a GyroLab xPlore instrument. The corresponding limit of quantitation (LLOQ) was defined as the lowest concentration on the standard curve whose quality control (QC) samples were determined to consistently deviate from the expected concentration by less than 25% (Table 5-12). Sample concentrations were determined by interpolation using a standard curve constructed using 4-parameter logic curve fitting in GyrolabEvaluator software. The final concentration was calculated using the average concentration from two replicate experiments.

[0500]

[0501] PK parameters were determined using non-compartmental analysis (NCA) with Phoenix® WinNonlin® software version 6.3 (Certara, Princeton, NJ, LP) and an extravascular administration model. All PK parameters were determined using the corresponding mean concentration value (total drug) for each antibody, employing a linear trapezoidal rule with linear interpolation and uniform weighting, incorporating the observed maximum concentration in serum (C0). max ), Observed estimated half-life (t) 1 / 2 ), area under the concentration curve comparison until the final measurable concentration (AUC) last) and antibody clearance rate (Cl).

[0502] 7.7.3. Results

[0503] In WT mice, after administration of the anti-TSLP Fc-Fab bispecific antibody at 1 mg / kg SC and the control, REGN8759 and REGN8760 showed similar maximum total drug concentrations in serum (C0.05, respectively). max = 11.7 and 10.7 μg / mL), while hIgG4 isotype control REGN1945, unrelated conventional bispecific IgG4 control H4H21237D, and hFcγ homodimer REGN1627 (C max The concentration of (dose normalized) decreased by approximately 1.5-2 times (C, respectively). max = 8.2, 7.8 and 6.4 μg / mL).

[0504] Furthermore, REGN8759, REGN8760, REGN1945, and H4H21237D all exhibit similar half-lives (T5, T6, T7, T8 ... 1 / 2 =12.1, 12.2, 10.9 and 11.2 days), while REGN1627 has a faster half-life (6.4 days) compared to all other tested drugs. Furthermore, compared to REGN1945, H4H21237D and REGN1627 (AUClast = 88.0, 84.1 and 19.7 respectively, AUClast = 88.0, 84.1 and 19.7 respectively), REGN1627 has a shorter half-life (6.4 days). last / D = 56.2 (d*μg / mL) / (mg / kg); Cl = 9.5, 8.2 and 45.2 mL / day / kg respectively), REGN8759 and REGN8760 showed better drug exposure (AUC respectively). last = 131 and 122 (d*μg / mL) / (mg / kg)) and slower clearance rates (Cl = 5.2 and 5.5 mL / day / kg, respectively).

[0505] Furthermore, the total functional TSLP binding concentrations of REGN8759 and REGN8760 were comparable across all test time points, indicating that these Fc-Fab molecules remained intact at 21 days. Overall, the PK curves of REGN8759 and REGN8760 were similar to or better than those of the hIgG4 isotype control, the unrelated conventional bispecific IgG4 control, or the hFcγ homodimer.

[0506] The total functional REGN8759 and REGN8760 drug concentrations, as well as the total REGN1945, H4H21237D, and REGN1627 drug concentrations, are summarized in Table 5-13. The average PK parameters are described in Table 5-14, and the average total antibody concentration over time is shown in [Table 5-14]. Figure 14 and Figure 15 middle.

[0507]

[0508]

[0509]

[0510] The PK parameter is derived from the average concentration-time curve of the total drug concentration. 1 / 2 and AUC last Based on the concentration on day 21, the mean ± SEM value of each PK parameter for all dose groups is shown.

[0511] Abbreviation: AUC last =Area under the curve from time of administration to final measurable concentration; AUC last / D = AUC normalized relative to 1 mg / kg dose; t½ = elimination half-life; C max =Peak concentration; C max / d = Cmax dose normalized relative to 1 mg / kg dosing; t max =Observed C max Time; Cl = antibody clearance rate over time; SEM = standard error of the mean.

[0512] 7.8. Example 8: Biacore analysis of the binding of anti-ligand X Fc-Fab to ligand X

[0513] Bispecific Fc-Fab for human ligand X was prepared using Fab fragments from three non-competitive mAbs targeting human ligand X, mAbX1, mAbX2, and mAbX3. Fc-Fab is a soluble monomeric protein with a molecular weight range of 15-20 kDa, wherein the linker is G4S2 (i.e., GGGGSGGGGS (SEQ ID NO: 18)). Fc-Fab possesses... Figure 13A The depiction of the hinge form.

[0514] The equilibrium dissociation constant (KD value) for the binding of ligand X to purified anti-ligand X antibody was determined using a real-time surface plasmon resonance biosensor on a Biacore T200 instrument. The Biacore sensor surface was derivatized by amine conjugation with a monoclonal mouse anti-human Fc antibody (REGN2567) to capture anti-ligand X antibody expressed in the human Fc constant region. Biacore binding studies were performed in HBST running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20). Human ligand X was obtained from an internal source (REGN138). Different concentrations of human ligand X (ranging from 90 nM to 0.12 nM, 3-fold dilutions) prepared in HBST running buffer were injected at a flow rate of 50 μL / min onto the surface captured by the anti-ligand X antibody. Binding of all ligand X reagents to each of the captured monoclonal antibodies was monitored for 4 minutes, and their dissociation in HBST run buffer was monitored for 10 minutes. All binding kinetics experiments were performed at 37°C. Kinetic association (ka) and dissociation (kd) rate constants were determined by fitting real-time sensor plots to a 1:1 binding model using dual-evaluation curve fitting software. The binding-dissociation equilibrium constant (KD) and dissociation half-life (t½) were calculated based on the kinetic rate constants as follows:

[0515] KD (M) = kd / ka and t½ (min) = ln2 / (60xkd)

[0516] The binding kinetics parameters of human ligand X to anti-ligand X antibody at 37 °C are shown in Table 5-15. At 37 °C, the anti-ligand X Fc-Fab of this disclosure binds to human ligand X with KD values ​​ranging from 0.25 pM to 18.4 pM, while the parental mAb binds to human ligand X with corresponding KD values ​​of 0.56 pM and 557 pM, as shown in Table 5-15.

[0517]

[0518] 7.9. Example 9: Activity of anti-human ligand X bispecific Fc-Fab in bioassays

[0519] The ability of purified anti-human ligand X bispecific Fc-Fab to inhibit human ligand X in receptor X signaling bioassays was evaluated. The bioassays of ligand X signaling via receptor X were performed using engineered luciferase reporter cell lines. Reporter cells were seeded at 10,000 cells / well in Opti-MEM (Gibco) containing 0.1% fetal bovine serum (Seradigm) and incubated overnight. For ligand X dose-response profiling, serially diluted ligand X was added to each well at a final concentration starting from 2 nM. To determine the blocking activity of the anti-ligand X parental mAb and the bispecific Fc-Fab, a “Race-form” blocking assay was used, in which the antibody and ligand X were simultaneously added to the reporter cells. The anti-ligand X antibody was serially diluted 1:3, starting at a final concentration of 100 nM. Human ligand X was added to constant concentrations of 10 pM, 100 pM, or 1 nM. After incubation for 5.5 hours, the assay plates were equilibrated at room temperature for 15 minutes. 100 μl of One-Glo substrate (Promega) was added to each well. After incubation at room temperature for 5 minutes, luminescence was measured on an Envision microscope.

[0520] The activity of three bispecific Fc-Fab anti-human ligand X in receptor X bioassays was shown Figures 16A-16C (mAbX1 x mAbX2) Figures 17A-17C (mAbX2 x mAbX3) and Figures 18A-18C (mAbX1 x mAbX3). Their activities were compared with the corresponding anti-human ligand X parental mAb in the same assay. Fc-Fab exhibited the best blocking activity, with IC50% higher than the parental anti-ligand X mAb. 50 The value was significantly improved. One of the parent mAbs, mAbX3, did not block the activity of ligand X against baseline in bioassays, even when the mAb was in 100 to 1000-fold molar excess of human ligand X. Interestingly, using both Fc-Fabs of mAbX3 was able to block the activity of ligand X against baseline. Figures 17A-17C and Figures 18A-18C These results indicate that the bispecific Fc-Fab exhibits higher activity compared to the parental anti-ligand X mAb. The IC50 values ​​of these anti-ligand X antibodies... 50 The values ​​are summarized in Table 5-16.

[0521]

[0522] 7.10. Example 10: Size analysis of in vitro complexes formed between antagonistic ligand X heterodimers and recombinant ligand X via asymmetric flow field coupled to multi-angle light scattering (A4F-MALLS).

[0523] Size analysis of in vitro complexes formed between recombinant ligands X in Fc-Fab, clamp and 2+2 tandem Fab heterodimer forms and bispecific mAbX1 xmAbX2 was performed relative to the complexes with the parent mAb using asymmetric flow field-flow fractionation coupled to multi-angle light scattering (A4F-MALLS), as described in Section 7.4. Figures 19A-19E The results of this analysis are shown. Figure 19A and 19B The in vitro analysis results of the complexes (alone or in combination) of ligand X and parent mAb are shown; Figure 19C The in vitro analysis results of the complex of ligand X with mAbX1 x mAbX2 Fc-Fab are shown; Figure 19D The in vitro analysis results of the clamp-type complexes of ligand X with mAbX1 and xmAbX2 are shown; and Figure 19E The in vitro analysis results of the complex of ligand X with the mAbX1 x mAbX2 2+2 tandem Fab heterodimer are shown. Figure 19C As shown, the Fc-Fab form displays a minimal amount of paper doll dressing up or gathering.

[0524] 7.11. Example 11: Fc-Fab maintains binding with cell surface targets

[0525] In addition to small soluble antigens, cell surface proteins were also tested as targets of Fc-Fab. The hIgG1 constant region ( Figure 20A or the hIgG4 constant region with reduced effector function (US9359437B2) (shown as hIgG4s, Figure 20B , 20C 20D) reformulates IgG antibodies against antigen Y (a cell surface antigen) into monospecific Fc-Fab (e.g., 20D). Figure 1B As shown, the hinge form is as follows Figure 13A(As depicted in the image). Three different adapters, 1xG4S (SEQ ID NO: 3), 2xG4S (SEQ ID NO: 18), and 3xG4S (SEQ ID NO: 4), were tested against each anti-antigen Y Fc-Fab. The binding of these Fc-Fabs to cell surface antigen Y was evaluated in a flow cytometry (FACS) binding assay. Cells expressing antigen Y were collected and resuspended in cold FACS wash buffer (PBS + 1% FBS). For each binding assay, 50,000–100,000 cells were incubated with the primary antibody in FACS wash buffer at 4°C for 30 min. The cells were then washed twice with cold FACS wash buffer and incubated at 4°C for 30 min with a 1:200 dilution of APC-F(ab)'2 anti-human IgG Fcγ fragment (Jackson Immuno Research Laboratories). At the end of the incubation, the cells were washed twice with cold FACS washing buffer and analyzed on FACS Canto (BD Biosciences).

[0526] All Fc-Fabs maintained strong binding to cell surface antigen Y. Both groups of Fc-Fabs exhibited binding activity similar to their parent mAbs. Figure 20A and 20B Compared to their parent mAbs, the other two Fc-Fab groups showed moderately reduced binding to antigen Y. Figure 20C and 20D The change in linker length between 1xG4S (SEQ ID NO: 3) and 3xG4S (SEQ ID NO: 4) has the least impact on target binding to the anti-antigen Y Fc-Fab.

[0527] Other cell surface proteins were tested as targets for Fc-Fab, including CD3 and cell surface tumor-associated antigen, antigen Z. Fc-Fab possesses... Figure 13A The hinge form depicted in the image. In FACS binding assays, anti-CD3 hIgG1 Fc-Fab showed specific binding to CD3+ Jurkat cells (…). Figure 21A ), while the anti-antigen Z hIgG1 Fc-Fab showed specific binding to the antigen Z+ cell line ( Figure 21B The variation in linker length between 1xG4S (SEQ ID NO: 3) and 5xG4S (SEQ ID NO: 39) has little effect on the target binding of these Fc-Fabs, with the shortest linker resulting in weaker binding activity to both CD3 and antigen Z.

[0528] 7.12. Example 12: Bispecific CD3 x antigen Z Fc-Fab is active in bioassays using T cells as effector cells.

[0529] As described in Example 1, a bispecific Fc-Fab targeting CD3 and antigen Z (cell surface tumor-associated antigen) is generated using the constant region of hIgG1. Figure 13A These bispecific Fc-Fabs, depicting hinge forms, were tested with three different adapters: 1xG4S (SEQ ID NO: 3), 2xG4S (SEQ ID NO: 18), and 3xG4S (SEQ ID NO: 4). In the Jurkat NFAT-luciferase reporter assay (… Figure 22A ) and in vitro cytotoxicity assays ( Figure 22B The activity of bispecific Fc-Fab was evaluated in the Jurkat NFAT-luciferase reporter assay. In the Jurkat / NFAT-Luc reporter cell line and the antigen Z+ cell line (50,000 cells per cell line) were mixed at a 1:1 ratio in 96-well plates. CD3 x antigen Z bispecific Fc-Fab was added to each well to a final volume of 100 μl. The reaction was incubated at 37 °C for 5 h. After incubation, the plate was equilibrated at room temperature for 10 min, and then 100 μl of One-Glo substrate (Promega) was added to each well. Luminescence was measured on a Victor. In the cytotoxicity assay, pre-activated human T cells were prepared using human donor PBMCs activated for 7 days with CD3 / CD28 beads and IL-2. On the day of the cytotoxicity assay, antigen Z+ cells were harvested and labeled with 8 μM calcein-AM (Invitrogen) for 30 min. Labeled target cells were washed twice and mixed with pre-activated human T cells at a ratio of 1:10, with approximately 10,000 target cells per well. Serial dilutions of CD3 x antigen Z bispecific Fc-Fab were added to a final volume of 200 μl. The reaction was incubated at 37°C for 3 hours. After incubation, the plate was centrifuged, and 100 μl of the supernatant was transferred to a translucent black clear plate for fluorescence reading. CD3 x antigen Z bispecific Fc-Fab was used in Jurkat reporter gene assays (…). Figure 22A ) and cytotoxicity assays ( Figure 22B Both assays showed activity. In both assays, Fc-Fab with a longer connector showed stronger activity. 8. Specific Implementation Examples

[0531] This disclosure is illustrated by the following specific embodiments from Group A and Group B.

[0532] In the preferred aspects of the following specific embodiments and subsequent claims, the antigen-binding domain (e.g., Fab) contains humanized or human VH and VL sequences; the Fc domain comprises human CH2 and / or CH3 domains and variants thereof, such as variants having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with such human sequences. Further, the Fab domain can be a Fab domain consisting of two polypeptide chains, as described herein, a VH polypeptide chain and a VL polypeptide chain, or a single-chain Fab (“scFab”) in which VH and VL are present in a single polypeptide chain. Unless otherwise explicitly stated, the Fab domain may also contain domain exchanges, such as those present in the Crossmab form.

[0533] 8.1. Specific Implementation Examples of Group A

[0534] 1. An antigen-binding molecule that binds to a first target molecule and:

[0535] (a) Includes:

[0536] (i) The Fc region, which includes two Fc structural domains;

[0537] (ii) First Fab domain and second Fab domain,

[0538] The Fc region, the first Fab domain, and the second Fab domain are in a non-natural immunoglobulin conformation.

[0539] Wherein the first Fab domain and / or the second Fab domain can bind to the first target molecule; and

[0540] (b) To bind the first target molecule with a greater affinity and / or affinity than natural immunoglobulins that include the at least two Fab domains.

[0541] 2. An antigen-binding molecule, optionally an antigen-binding molecule according to Example 1, said antigen-binding molecule binding to a first target molecule and comprising:

[0542] (a) A first polypeptide, wherein the first polypeptide comprises, in the N-to-C-terminal orientation:

[0543] (i) The first Fc structural domain; and

[0544] (ii) a first Fab domain, the first Fab domain comprising a first heavy chain variable region (VH) associated with a first light chain variable region (VL); and

[0545] (b) A second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0546] (i) The second Fc structural domain; and

[0547] (ii) A second Fab domain, the second Fab domain comprising a second VH associated with a second VL,

[0548] The first Fc domain and the second Fc domain associate with each other to form an Fc region, and optionally the first polypeptide and the second polypeptide are the same.

[0549] 3. The antigen-binding molecule according to Example 2, comprising a first linker between the first Fc domain and the first VH.

[0550] 4. The antigen-binding molecule according to Example 3, wherein the length of the first linker is from 5 to 60 amino acids.

[0551] 5. The antigen-binding molecule according to Example 3, wherein the length of the first linker is from 10 to 60 amino acid residues.

[0552] 6. The antigen-binding molecule according to Example 3, wherein the length of the first linker is from 5 to 20 amino acid residues.

[0553] 7. The antigen-binding molecule according to Example 3, wherein the length of the first linker is from 5 to 30 amino acid residues.

[0554] 8. The antigen-binding molecule according to Example 3, wherein the length of the first linker is 10 to 30 amino acid residues.

[0555] 9. The antigen-binding molecule according to Example 3, wherein the length of the first linker is 10 to 20 amino acid residues.

[0556] 10. The antigen-binding molecule according to Example 3, wherein the length of the first linker is 20 to 50 amino acids.

[0557] 11. The antigen-binding molecule according to Example 3, wherein the length of the first linker is 25 to 35 amino acids.

[0558] 12. The antigen-binding molecule according to any one of Examples 3 to 11, wherein the first linker comprises G n S or SG n The polymer of , optionally where n is an integer from 1 to 7.

[0559] 13. The antigen-binding molecule according to Example 12, wherein the first linker comprises a polymer of G4S (SEQ ID NO:3).

[0560] 14. The antigen-binding molecule according to Example 13, wherein the first linker comprises 2 to 6 repeats of G4S (SEQ ID NO:3).

[0561] 15. The antigen-binding molecule according to Example 14, wherein the first linker comprises (G4S)2 (SEQ ID NO: 18), (G4S)3 (SEQ ID NO: 4), or (G4S)4 (SEQ ID NO: 19).

[0562] 16. The antigen-binding molecule according to any one of Examples 3 to 15, comprising a second linker between the second Fc domain and the second VH.

[0563] 17. The antigen-binding molecule according to Example 16, wherein the first adapter and the second adapter have the same amino acid sequence.

[0564] 18. The antigen-binding molecule according to Example 16 or Example 17, wherein the length of the second linker is from 5 amino acids to 60 amino acids.

[0565] 19. The antigen-binding molecule according to Example 16 or Example 17, wherein the length of the second linker is from 10 to 60 amino acids.

[0566] 20. The antigen-binding molecule according to Example 16 or Example 17, wherein the length of the first linker is from 5 to 20 amino acid residues.

[0567] 21. The antigen-binding molecule according to Example 16 or Example 17, wherein the length of the first linker is from 5 to 30 amino acid residues.

[0568] 22. The antigen-binding molecule according to Example 16 or Example 17, wherein the length of the first linker is 10 to 30 amino acid residues.

[0569] 23. The antigen-binding molecule according to Example 16 or Example 17, wherein the length of the first linker is 10 to 20 amino acid residues.

[0570] 24. The antigen-binding molecule according to Example 16 or Example 17, wherein the length of the second linker is 20 to 50 amino acids.

[0571] 25. The antigen-binding molecule according to Example 16 or Example 17, wherein the length of the second linker is 25 to 35 amino acids.

[0572] 26. The antigen-binding molecule according to any one of Examples 16 to 25, wherein the second linker comprises G n S or SG n The polymer of , optionally where n is an integer from 1 to 7.

[0573] 27. The antigen-binding molecule according to Example 26, wherein the second linker comprises a polymer of G4S (SEQ ID NO:3).

[0574] 28. The antigen-binding molecule according to Example 27, wherein the second linker comprises 2 to 6 repeats of G4S (SEQ ID NO:3).

[0575] 29. The antigen-binding molecule according to Example 28, wherein the second linker comprises (G4S)2 (SEQ ID NO: 18), (G4S)3 (SEQ ID NO: 4), or (G4S)4 (SEQ ID NO: 19).

[0576] 30. The antigen-binding molecule according to any one of Examples 2 to 29, wherein the first polypeptide includes a first hinge domain located at the N-terminus of the first Fc domain and the second polypeptide includes a second hinge domain located at the N-terminus of the second Fc domain.

[0577] 31. The antigen-binding molecule according to Example 30, wherein the first hinge domain and the second hinge domain are connected by disulfide bonds.

[0578] 32. The antigen-binding molecule according to Example 30, wherein the first hinge domain and the second hinge domain are not connected by disulfide bonds.

[0579] 33. The antigen-binding molecule according to any one of Examples 2 to 32, wherein the first polypeptide does not include VH located at the N-terminus of the first Fc domain.

[0580] 34. The antigen-binding molecule according to any one of Examples 2 to 33, wherein the second polypeptide does not include VH located at the N-terminus of the second Fc domain.

[0581] 35. The antigen-binding molecule according to any one of Examples 2 to 29, having a hinge region.

[0582] 36. The antigen-binding molecule according to Example 35, which has Figure 13A The hinge type shown.

[0583] 37. The antigen-binding molecule according to Example 35, which has Figure 13C The hinge type shown.

[0584] 38. The antigen-binding molecule according to any one of Examples 2 to 29, having two hinge regions.

[0585] 39. The antigen-binding molecule according to Example 38, which has Figure 13B The hinge type shown.

[0586] 40. The antigen-binding molecule according to any one of Examples 2 to 39, wherein the first polypeptide and the second polypeptide are not the same.

[0587] 41. The antigen-binding molecule according to any one of Examples 2 to 40, wherein the first VL and the second VL are universal light chains.

[0588] 42. The antigen-binding molecule according to any one of Examples 2 to 40, wherein the light chain constant region and the first heavy chain constant region (CH1) of the first Fab domain or the second Fab domain are arranged in a crossmab configuration.

[0589] 43. The antigen-binding molecule according to any one of Examples 1 to 42 is divalent.

[0590] 44. An antigen-binding molecule, optionally an antigen-binding molecule according to Example 1, said antigen-binding molecule comprising:

[0591] (a) A first polypeptide, wherein the first polypeptide comprises, in the N-to-C-terminal orientation:

[0592] (i) A first Fab domain, the first Fab domain including a first VH associated with a first VL;

[0593] (ii) First spaced structural domain;

[0594] (iii) The first Fc structural domain; and

[0595] (b) A second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0596] (i) A second Fab structural domain, the second Fab structural domain including a second VH associated with a second VL;

[0597] (ii) Second spaced structural domain;

[0598] (iii) The second Fc structural domain; and

[0599] The first Fab domain and / or the second Fab domain are capable of binding to the first target molecule, wherein the first Fc domain and the second Fc domain associate with each other to form an Fc region, and optionally wherein the first polypeptide and the second polypeptide are identical.

[0600] 45. The antigen-binding molecule according to Example 44, comprising a hinge domain between the first spacer domain and the first Fc domain and between the second spacer domain and the second Fc domain.

[0601] 46. ​​The antigen-binding molecule according to Example 44 or Example 45, wherein the hinge domains are connected by disulfide bonds.

[0602] 47. The antigen-binding molecule according to any one of Examples 44 to 46, wherein the first VL and the second VL are universal light chains.

[0603] 48. The antigen-binding molecule according to any one of Examples 44 to 46, wherein the light chain constant region and the first heavy chain constant region (CH1) of the first Fab domain or the second Fab domain are arranged in a crossmab configuration.

[0604] 49. The antigen-binding molecule according to any one of Examples 44 to 48, wherein the first spacer domain and the second spacer domain each include an extension linker.

[0605] 50. The antigen-binding molecule according to Example 49, wherein each extension linker is at least 30 amino acids in length.

[0606] 51. The antigen-binding molecule according to Example 49 or Example 50, wherein each extension linker is 30 acid residues to 70 amino acids in length.

[0607] 52. The antigen-binding molecule according to Example 49 or Example 50, wherein each extension linker is 30 acid residues to 55 amino acids in length.

[0608] 53. The antigen-binding molecule according to Example 49 or Example 50, wherein each extension linker is 30 acid residues to 40 amino acids in length.

[0609] 54. The antigen-binding molecule according to any one of Examples 49 to 53, wherein each extension linker includes G n S or SG n The polymer of , optionally where n is an integer from 1 to 7.

[0610] 55. The antigen-binding molecule according to Example 54, wherein each extension linker comprises a polymer of G4S (SEQ ID NO:3).

[0611] 56. The antigen-binding molecule according to Example 55, wherein each extension linker comprises 5 to 12 repeats of G4S (SEQ ID NO:3).

[0612] 57. The antigen-binding molecule according to Example 56, wherein each extension linker comprises (G4S)6 (SEQ ID NO: 38), (G4S)7 (SEQ ID NO: 36), or (G4S)8 (SEQ ID NO: 37).

[0613] 58. The antigen-binding molecule according to any one of Examples 44 to 57, wherein the first and second spacer domains are identical.

[0614] 59. The antigen-binding molecule according to any one of Examples 44 to 58 is divalent.

[0615] 60. The antigen-binding molecule according to Example 44, wherein the first spacer domain includes a third Fab domain, the third Fab domain includes a third VH associated with a third VL, and the second spacer domain includes a fourth Fab domain, the fourth Fab domain including a fourth VH associated with a fourth VL.

[0616] 61. The antigen-binding molecule according to Example 60, wherein the third VL and the fourth VL are universal light chains, wherein the Fc region is [missing information].

[0617] 62. The antigen-binding molecule according to Example 60 or Example 61, wherein the first polypeptide includes a first linker between the first VH and the third VH, and the second polypeptide includes a second linker between the second VH and the fourth VH.

[0618] 63. The antigen-binding molecule according to Example 62, wherein the length of the first linker and the second linker is each from 10 amino acids to 60 amino acids.

[0619] 64. The antigen-binding molecule according to Example 62 or Example 63, wherein the length of the first linker and the second linker is each 20 to 50 amino acids.

[0620] 65. The antigen-binding molecule according to Example 62 or Example 63, wherein the length of the first linker and the second linker is 25 to 35 amino acids.

[0621] 66. The antigen-binding molecule according to Example 62 or Example 63, wherein the first adapter and the second adapter each comprise G n S or SG n The polymer of , optionally where n is an integer from 1 to 7.

[0622] 67. The antigen-binding molecule according to Example 66, wherein the first adapter and the second adapter each comprise a polymer of G4S (SEQ ID NO: 3).

[0623] 68. The antigen-binding molecule according to Example 67, wherein the first adapter and the second adapter each comprise 2 to 6 repeats of G4S (SEQ ID NO: 3).

[0624] 69. The antigen-binding molecule according to Example 68, wherein the first adapter and the second adapter each comprise (G4S)2 (SEQ ID NO: 18), (G4S)3 (SEQ ID NO: 4), or (G4S)4 (SEQ ID NO: 19).

[0625] 70. The antigen-binding molecule according to any one of Examples 62 to 69, wherein the first adapter and the second adapter have the same amino acid sequence.

[0626] 71. The antigen-binding molecule according to any one of Examples 60 to 70, wherein the third Fab domain and the fourth Fab domain are non-binding.

[0627] 72. The antigen-binding molecule according to Example 71, wherein the third VH and the fourth VH are universal heavy chains.

[0628] 73. The antigen-binding molecule according to Example 71 or Example 72, wherein the first Fab domain and the second Fab domain are each capable of binding the same or different epitopes on the first target molecule.

[0629] 74. The antigen-binding molecule according to any one of Examples 60 to 73 is bivalent.

[0630] 75. The antigen-binding molecule according to any one of Examples 60 to 70, wherein the third Fab domain and the fourth Fab domain are each capable of binding the same or different epitopes.

[0631] 76. The antigen-binding molecule according to Example 75, wherein the third Fab domain and the fourth Fab domain are each capable of binding the same target molecule.

[0632] 77. The antigen-binding molecule according to Example 76, wherein the third Fab domain and the fourth Fab domain are each capable of binding the first target molecule.

[0633] 78. The antigen-binding molecule according to any one of Examples 75 to 77, wherein the first and second Fab domains bind to the same epitope.

[0634] 79. The antigen-binding molecule according to Example 78, wherein the first and second Fab domains have the same sequence.

[0635] 80. The antigen-binding molecule according to any one of Examples 75 to 79, wherein the third and fourth Fab domains bind the same epitope.

[0636] 81. The antigen-binding molecule according to Example 80, wherein the third and fourth Fab domains have the same sequence.

[0637] 82. The antigen-binding molecule according to any one of Examples 75 to 77, wherein the first and third Fab domains bind the same epitope.

[0638] 83. The antigen-binding molecule according to Example 82, wherein the first and third Fab domains have the same sequence.

[0639] 84. The antigen-binding molecule according to any one of Examples 75 to 77, 82 and 83, wherein the second and fourth Fab domains bind the same epitope.

[0640] 85. The antigen-binding molecule according to Example 84, wherein the second and fourth Fab domains have the same sequence.

[0641] 86. The antigen-binding molecule according to any one of Examples 60 to 70 and 75 to 85 is tetravalent.

[0642] 87. The antigen-binding molecule according to any one of Examples 1 to 86, which is an antagonist of the first target molecule.

[0643] 88. The antigen-binding molecule according to any one of Examples 1 to 87, which inhibits the binding of the first target molecule to a binding partner, wherein optionally the binding partner is a receptor for the first target molecule.

[0644] 89. The antigen-binding molecule according to any one of Examples 1 to 88, wherein the Fc region comprises a human Fc sequence.

[0645] 90. The antigen-binding molecule according to any one of Examples 1 to 89, wherein the Fc region comprises a human IgG1 or human IgG4 Fc sequence.

[0646] 91. The antigen-binding molecule according to any one of Examples 1 to 90, wherein the Fc region comprises an Fc heterodimer.

[0647] 92. The antigen-binding molecule according to Example 89, wherein the Fc domain in the Fc heterodimer includes an intrapore toggle mutation compared to the wild-type Fc domain.

[0648] 93. The antigen-binding molecule according to Example 92, wherein the Fc domain of the first polypeptide includes a button mutation and the Fc domain of the second polypeptide includes a pore mutation.

[0649] 94. The antigen-binding molecule according to Example 92, wherein the Fc domain of the second polypeptide includes a button mutation and the Fc domain of the first polypeptide includes a pore mutation.

[0650] 95. The antigen-binding molecule according to Example 89, wherein the Fc region includes a star mutation compared to the wild-type Fc region.

[0651] 96. The antigen-binding molecule according to Example 89, wherein the Fc domain of the first polypeptide includes the H435R mutation and the Y436F mutation.

[0652] 97. The antigen-binding molecule according to Example 89, wherein the Fc domain of the second polypeptide includes the H435R mutation and the Y436F mutation.

[0653] 98. The antigen-binding molecule according to any one of Examples 1 to 97, wherein the CL and CH1 in the first Fab domain are linked by a disulfide bond.

[0654] 99. The antigen-binding molecule according to any one of Examples 1 to 98, wherein the CL and CH1 in the second Fab domain are linked by disulfide bonds.

[0655] 100. The antigen-binding molecule according to any one of Examples 1 to 99, wherein the first Fab domain and the second Fab domain bind to the first target molecule.

[0656] 101. The antigen-binding molecule according to any one of Examples 1 to 100, wherein the first target molecule is a small soluble ligand.

[0657] 102. The antigen-binding molecule according to any one of Examples 1 to 101, wherein the first target molecule is a cytokine or chemokine.

[0658] 103. The antigen-binding molecule according to any one of Examples 1 to 100, wherein the first target molecule is a cell surface protein.

[0659] 104. The antigen-binding molecule according to any one of Examples 1 to 100 and 103, wherein the first target molecule is a tumor-associated antigen.

[0660] 105. The antigen-binding molecule according to any one of Examples 1 to 104, wherein the first target molecule has a molecular weight of less than 100 kDa and does not contain post-translational modifications.

[0661] 106. The antigen-binding molecule according to any one of Examples 1 to 104, wherein the first target molecule has a molecular weight of less than 100 kDa and includes post-translational modifications.

[0662] 107. The antigen-binding molecule according to any one of Examples 1 to 104, wherein the first target molecule has a molecular weight of less than 75 kDa and does not contain post-translational modifications.

[0663] 108. The antigen-binding molecule according to any one of Examples 1 to 104, wherein the first target molecule has a molecular weight of less than 75 kDa and includes post-translational modifications.

[0664] 109. The antigen-binding molecule according to any one of Examples 1 to 104, wherein the first target molecule has a molecular weight of less than 60 kDa and does not contain post-translational modifications.

[0665] 110. The antigen-binding molecule according to any one of Examples 1 to 104, wherein the first target molecule has a molecular weight of less than 60 kDa and includes post-translational modifications.

[0666] 111. The antigen-binding molecule according to any one of Examples 1 to 104, wherein the first target molecule has a molecular weight of less than 45 kDa and does not contain post-translational modifications.

[0667] 112. The antigen-binding molecule according to any one of Examples 1 to 104, wherein the first target molecule has a molecular weight of less than 45 kDa and includes post-translational modifications.

[0668] 113. The antigen-binding molecule according to any one of Examples 1 to 112, wherein the first target molecule has a molecular weight of at least 5 kDa and does not contain post-translational modifications.

[0669] 114. The antigen-binding molecule according to any one of Examples 1 to 112, wherein the first target molecule has a molecular weight of at least 5 kDa and includes post-translational modifications.

[0670] 115. The antigen-binding molecule according to any one of Examples 1 to 112, wherein the first target molecule has a molecular weight of at least 5 kDa and does not contain post-translational modifications.

[0671] 116. The antigen-binding molecule according to any one of Examples 1 to 112, wherein the first target molecule has a molecular weight of at least 5 kDa and includes post-translational modifications.

[0672] 117. The antigen-binding molecule according to any one of Examples 1 to 112, wherein the first target molecule has a molecular weight of at least 10 kDa and does not contain post-translational modifications.

[0673] 118. The antigen-binding molecule according to any one of Examples 1 to 112, wherein the first target molecule has a molecular weight of at least 10 kDa and includes post-translational modifications.

[0674] 119. The antigen-binding molecule according to any one of Examples 1 to 118, wherein the first target molecule is glycosylated.

[0675] 120. The antigen-binding molecule according to any one of Examples 1 to 118, wherein the first target molecule is not glycosylated.

[0676] 121. The antigen-binding molecule according to any one of Examples 1 to 120, wherein the first target molecule is a monomer.

[0677] 122. The antigen-binding molecule according to any one of Examples 1 to 120, wherein the first target molecule is a dimer.

[0678] 123. The antigen-binding molecule according to Example 122, wherein the first target molecule is a homodimer.

[0679] 124. The antigen-binding molecule according to Example 122, wherein the first target molecule is a heterodimer.

[0680] 125. The antigen-binding molecule according to any one of Examples 1 to 120, wherein the first target molecule is a trimer.

[0681] 126. The antigen-binding molecule according to Example 125, wherein the first target molecule is a homotrimer.

[0682] 127. The antigen-binding molecule according to any one of Examples 1 to 120, wherein the first target molecule is a tetramer.

[0683] 128. The antigen-binding molecule according to Example 127, wherein the first target molecule is a homotetramer.

[0684] 129. The antigen-binding molecule according to any one of Examples 1 to 128 is single-specific.

[0685] 130. The antigen-binding molecule according to any one of Examples 1 to 128 is bispecific.

[0686] 131. The antigen-binding molecule according to Example 130, which is capable of binding to the first epitope and the second epitope on the first target molecule.

[0687] 132. The antigen-binding molecule according to Example 131, comprising at least one Fab domain binding to the first epitope on the first target molecule and at least one Fab domain binding to the second epitope.

[0688] 133. The antigen-binding molecule according to Example 132 is capable of simultaneously binding to different epitopes on the first target molecule.

[0689] 134. The antigen-binding molecule according to Example 130, which is capable of binding the first target molecule and binding the second target molecule.

[0690] 135. The antigen-binding molecule according to Example 134, comprising at least one Fab domain binding the first target molecule and at least one Fab domain binding the second target molecule.

[0691] 136. The antigen-binding molecule according to Example 135 can bind to both the first target molecule and the second target molecule simultaneously.

[0692] 137. The antigen-binding molecule according to any one of Examples 1 to 136, relative to a human IgG antibody comprising the first Fab and the second Fab, exhibits a lower IC50 value. 50 Blocking the binding of the target molecule to its receptor.

[0693] 138. The antigen-binding molecule according to any one of Examples 1 to 137 binds the target molecule with greater affinity than a human IgG antibody comprising the first Fab and the second Fab.

[0694] 139. A conjugate comprising an antigen-binding molecule according to any one of Examples 1 to 138 and a cytotoxic agent or cell inhibitor.

[0695] 140. A pharmaceutical composition comprising an antigen-binding molecule according to any one of Examples 1 to 138 or a conjugate according to Example 139 and an excipient.

[0696] 141. A method of treating a subject suffering from symptoms associated with abnormal expression or activity of a target molecule, the method comprising administering to the subject an effective amount of an antigen-binding molecule according to any one of Examples 1 to 138, a conjugate according to Example 139, or a pharmaceutical composition according to Example 140.

[0697] 142. A method for inhibiting molecular pathways associated with a target molecule in a subject, the method comprising administering to the subject an effective amount of an antigen-binding molecule according to any one of Examples 1 to 138, a conjugate according to Example 139, or a pharmaceutical composition according to Example 140.

[0698] 143. Use in the preparation of a medicament of an antigen-binding molecule according to any one of Examples 1 to 138, a conjugate according to Example 139, or a pharmaceutical composition according to Example 140, for treating symptoms associated with a target molecule bound by the antigen-binding molecule, the conjugate, or the antigen-binding molecule or conjugate present in the pharmaceutical composition, respectively.

[0699] 144. A nucleic acid molecule or multiple nucleic acid molecules comprising one or more nucleotide sequences encoding an antigen-binding molecule according to any one of Examples 1 to 138.

[0700] 145. The nucleic acid molecule or multiple nucleic acid molecules according to Example 144, wherein one or more nucleotide sequences are each operatively linked to an expression control sequence.

[0701] 146. A cell engineered to express an antigen-binding molecule according to any one of Examples 1 to 138.

[0702] 147. A cell transfected with one or more expression vectors, said expression vectors comprising one or more nucleic acid sequences encoding an antigen-binding molecule according to any one of Examples 1 to 138 under the control of one or more promoters.

[0703] 148. A method for generating an antigen-binding molecule according to any one of Examples 1 to 138, the method comprising:

[0704] (a) Culturing cells according to Example 146 or Example 147 under conditions expressing the antigen-binding molecule; and

[0705] (b) Recovery of the antigen-binding molecule from the cell culture

[0706] 149. The method according to Example 148, further comprising enriching the antigen-binding molecule.

[0707] 150. The method according to Example 148 or Example 149, further comprising purifying the antigen-binding molecule.

[0708] 8.2. Specific Implementation Examples of Group B

[0709] 1. An antigen-binding molecule comprising:

[0710] (a) A first heavy chain polypeptide comprising: a first CH amino acid sequence; a first VH amino acid sequence; and a second CH amino acid sequence, wherein the first VH amino acid sequence is located between the first CH amino acid sequence and the second CH amino acid sequence; and

[0711] (b) A second heavy chain polypeptide comprising: a third CH amino acid sequence; a second VH amino acid sequence; and a fourth CH amino acid sequence, wherein the second VH amino acid sequence is located between the third CH amino acid sequence and the fourth CH amino acid sequence.

[0712] 2. The antigen-binding molecule according to Example 1, wherein the first CH amino acid sequence includes a first CH3 amino acid sequence located at the N-terminus of the first VH amino acid sequence.

[0713] 3. The antigen-binding molecule according to Example 2, wherein the first CH3 includes the H435R mutation and the Y436F mutation.

[0714] 4. The antigen-binding molecule according to Example 2 or Example 3, further comprising a first adapter connecting the N-terminus of the first VH amino acid sequence to the C-terminus of the first CH3 amino acid sequence.

[0715] 5. The antigen-binding molecule according to any one of Examples 1 to 4, wherein the third CH amino acid sequence comprises a second CH3 amino acid sequence located at the N-terminus of the second VH amino acid sequence.

[0716] 6. The antigen-binding molecule according to Example 5, further comprising a second adapter connecting the N-terminus of the second VH amino acid sequence to the C-terminus of the second CH3 amino acid sequence.

[0717] 7. The antigen-binding molecule according to any one of Examples 1 to 6, wherein the second CH amino acid sequence comprises a first CH1 amino acid sequence located at the C-terminus of the first VH amino acid sequence.

[0718] 8. The antigen-binding molecule according to any one of Examples 1 to 7, wherein the fourth CH amino acid sequence comprises a second CH2 amino acid sequence located at the C-terminus of the second VH amino acid sequence.

[0719] 9. The antigen-binding molecule according to any one of Examples 1 to 8, further comprising a first CH2 amino acid sequence located at the N-terminus of the first CH3 amino acid sequence.

[0720] 10. The antigen-binding molecule according to any one of Examples 1 to 9, further comprising a second CH2 amino acid sequence located at the N-terminus of the second CH3 amino acid sequence.

[0721] 11. The antigen-binding molecule according to any one of Examples 1 to 10, wherein the antigen-binding molecule does not contain hinge region disulfide bonds.

[0722] 12. The antigen-binding molecule according to any one of Examples 1 to 11, further comprising a first light chain polypeptide, the first light chain polypeptide comprising: a first VL amino acid sequence; and a first CL amino acid sequence.

[0723] 13. The antigen-binding molecule according to Example 12, further comprising a disulfide bond connecting the first CL to the first CH1.

[0724] 14. The antigen-binding molecule according to any one of Examples 1 to 13, further comprising a second light chain polypeptide, the second light chain polypeptide comprising: a second VL amino acid sequence; and a second CL amino acid sequence.

[0725] 15. The antigen-binding molecule according to Example 14, further comprising a disulfide bond connecting the second CL to the second CH1.

[0726] 16. The antigen-binding molecule according to any one of Examples 6 to 15, wherein the first adapter and the second adapter each comprise a polypeptide.

[0727] 17. The antigen-binding molecule according to Example 16, wherein the first linker and the second linker have a length of 0 to 50 amino acids.

[0728] 18. The antigen-binding molecule according to any one of Examples 16 to 17, wherein the first adapter and the second adapter have the same amino acid sequence.

[0729] 19. The antigen-binding molecule according to any one of Examples 16 to 18, wherein the first adapter and the second adapter each comprise a polyglycine and a serine amino acid sequence.

[0730] 20. The antigen-binding molecule according to Example 19, wherein the polyglycine and serine amino acid sequences comprise 2 to 6 repeating GGGGS (SEQ ID NO: 3) amino acid sequences.

[0731] 21. The antigen-binding molecule according to Example 20, wherein the polyglycine and serine amino acid sequences comprise (G4S)2 (SEQ ID NO: 18), (G4S)3 (SEQ ID NO: 4), or (G4S)4 (SEQ ID NO: 19).

[0732] 22. The antigen-binding molecule according to any one of Examples 14 to 21, wherein the first light chain polypeptide and the second light chain polypeptide have the same amino acid sequence.

[0733] 23. The antigen-binding molecule according to any one of Examples 1 to 22, wherein the first heavy chain polypeptide and the second heavy chain polypeptide have the same amino acid sequence.

[0734] 24. The antigen-binding molecule according to any one of Examples 1 to 22, wherein the first heavy chain polypeptide and the second heavy chain polypeptide have different amino acid sequences.

[0735] 25. The antigen-binding molecule according to any one of Examples 1 to 24, wherein the antigen-binding molecule is capable of binding one or more antigens selected from the group consisting of: ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVR1I, ADORA2A, Aggrecan, AGR2, AICDA, AIF1, AIG1, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, AZGP1 (zinc-α-glycoprotein), ART-4, B7, B7.1, B7.2, BAD, BAFF, BAGI, BAIi, BCL2, BCL6, BDNF, BLNK, BLRl (MDRlS), BlyS, BMPl, BMP2, BMP3B (GDF10), BMP4, BMP6, BMPS, BMPR1A, BMPR1B, BMPR2, BPAG1 (lectin), BRCA1, Ba -733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-I, CASP-8 / m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CDS, CD8, CDI-IA, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD8 0. CD83, CD95, CD126, CD133, CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CXCR4, CXCR7, CXCL12, C19orf10 (IL27w), C3, C4A, CS, CSR1, CANT1, CASPI, CASP4, CAV1, CCBP2 (D6 / JAB61), CCLI (I-309), CCLII (chemokine), CCL13 (MCP-4), CCLIS (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCLIS (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MIP-2), SLC, exodus-2, CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / chemokine-2), CCL2S (TECK)CCL26 (chemokine-3), CCL27 (CTACK / ILC), CCL2S, CCL3 (MIP1a), CCL4 (MIP-1b), CCLS (RANTES), CCL7 (MCP-3), CCLS (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1 / HM14S), CCR2 (mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCRS (CMKBRSIChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EB1), CCRS (CMKBRS / TER1 / CKR-LI), CCR9 (GPR-9-6), CCRLI (VSHK1) CCRL2 (L-CCR), CD164, CDlC, CD200, CD-22, CD24, CD2S, CD3S, CD3E, CD3G, CD3Z, CD4, CD44, C D4SRB, CD47, CD4S, CDS2, CD69, CD72, CD79A, CD79B, CDSO, CDS1, CDS3, CDS6, CD137, CD13S, B7 -1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CDH1 (E-cadherin), CDH10, CDH12, CDH13, CDHlS, CDH19, CDH20, CDHS, CDH7, CDHS, CDH9, CDK2, CDK3, CDK4, CDKS, CDK6, CDK7, CDK9, CDKN1A (p21 Wap1 / Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEBPB, CER1, CHGA, CHGB, chitinase, CHST1O, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSFS, CKLFSF6, CKLFSF7, CKLFSFS, CLDN3, CLDN7 (blocking-7), CLN3, CLU (polybutase) Cluster proteins), CMKLR1, CMKOR1 (RDC1), CNR1, COLISA1, COLIA1, COL4A3, COL6A1, CR2, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA-4, CTNNB1 (β-catenin), CTSB (cathepsin B), CX3CLI (SCYD1), CX3CR1 (V2S), CXCLI (GRO1), CXCLIO (IP-10)CXCL11 (I-TAC / IP-9), CXCL13, CXCL14, CXCL16, CXCL2 (GR02), CXCL3 (GR03), CXCLS (ENA-7S / LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), CYBS, CYC1, CYSLTR1, HIF-1-a, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-met, DAB2IP, DES, DKFZp4S1J01 1S, DNCLI, DPP4, DAM, EGFR, EGFRvllll, EGP-1, EGP-2, ELF2-M, Ep-CAM, E2F1, ECGF1, EDG1, EFNA1, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, EN01, EN02, E N03, EPHB4, EPO, EREG, ERKS, ESR1, ESR2, F3 (TF), FADD, FasL, FASN, FCER1A, FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF1S, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGFS, FGF7 (KGF), FGFS, FGF9, FGFR3, FIGF (VEGFD), FILI (EPSILON), FILI (ZETA), FLJ12SS4, FLJ2SS30, FLRT1 (fibronectin), FOS, FOSLI (FRA-1), FY (DARC), Flt-I, Flt-3, folate receptor, G250 antigen, GAGE, GROB GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GDFS, GFil, GGT1, GM-CSF, GNAS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPRS1 (FKSGSO), GRCC10 (C10), GRP, GSN (gelatin), GSTP1, HAVCR2, HDAC4, HDACS, HDAC7A, HDAC9, HGF, HIP1 histamine and histamine receptor, HLA-A, HLA-DRA, HM74, HMOX1, HUMCYT2A, HLA-DR, HMI 24, human chorionic gonadotropin (HCG) and its subunits,HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2 or 1a, IGF-IR, IFN-ɣ, IFN-α, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IGBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-1, IL-10, IL-10RA, IL-10RB, IL-11, IL-11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL-13RA1, IL-13RA2, IL-14, IL-1S, IL-1SRA, IL-16, IL-17, IL-17B, IL-17C, IL-17R, IL-18, IL-18BP, IL-18R1, IL-18RAP, IL-19, IL-IA, IL-1B, IL-1F10, IL-1FS, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-1HY1, IL-1R1, IL-1R2, IL-1RAP, IL-1RAPL1, IL-1RAPL2, IL-1RL1, IL-1RL2 IL-1RN, IL-2, IL-20, IL-20RA, IL-21R, IL-22, IL-22R, IL-22RA2, IL-23, IL-24, IL-2S, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-2RA, IL-2RB, IL-2RG, IL-3, IL-30, IL-3RA, IL-4, IL-4R, IL-S, IL-5RA, IL-6, IL-6R, IL-6ST (glycoprotein 130), IL-7, IL-7R, IL-S, IL-SRA, IL-SRB, IL-9, IL-9R, IL-K, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (a6 antigen), ITGAV, ITGB3, ITGB4 (b4 integrin), insulin-like growth factor-I (IGF-1), ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4IFNAS, IFNA6, IFNA7, IFNB1, IFNW1, JAG1, JAK1, JAK3, JUN, K6HF, KAi1, KDR, KITLG, KLFS (GC box BP), KLF6, KLK10, KLK12, KLK13KLK14, KLK1S, KLK3, KLK4, KLKS, KLK6, KLK9, KRT1, KRT19 (keratin 19), KRT2A, KRTHB6 (hair-specific type II keratin), KC4-antigen, KS-1-antigen, KS 1-4, Le-Y, LDR / FUT, LAMAS, LEP (leptin), Lingo-p7S, Lingo-Troy, LPS, LTA (TNF-β), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​MACMARCKS, MAG or Omgp, MAP2K7 (c-Jun), MDK, MIB1, intermediate factor, MIF, MIP-2, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionein-III), MTSS1, MUC1 (mucin), MYC, M YD88, Macrophage Migration Inhibitor Factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, NCK2, Neuroenergies, NFKB1, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgR-Nogo66 (Noga), NgRp7S, NgR- Troy, NME1 (NM23A), NOXS, NPPB, NROB1, NROB2, NR1D1, NR1D2, NRIH2, NRIH3, NRIH4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NRSA1, NRSA2, NR6A1, NRP1, NRP2, NTSE, NTN4, ODZ1, OPRD1, PCSK9, P2RX7, PAP, PART1 PATE, PAWR, PCA3, PCNA, PD-1, PD-L1, α4β7, OX40, GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDFS, CGRP, Lingo-I, Factor IXa, Factor X, ICOS, GARP, BTLA, CD160, RORI, 2B4, KIR, CD27, OX40, A2aR, PDGFA, PDGFB, PECAM1, PF4 (CXCL4), PGF, PGR, phosphatidylcholine, PIAS2, PIK3CG, PLAU (uPA), PLG, PLXDC1PPBP (CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2 (COX-2), PTN, pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, 10 PIGF, ILGF, ILGF-IR, IL-6, RS5, RANTES, RAC2 (p21Rac2), RARB, RGS1, RGS13, RGS3, RNF110 (ZNF144), ROB02, S100A2, SCGB1D2 (lipophilic B), SCGB2A1 (mammary globin 2), SCGB2A2 (mammary globin 1), SCYE1 (endothelial monocyte activation cytokine), SDF2, SERPINA1, SERPINA3, SERPINABS (mammary filament inhibitory protein), SERPINE1 (P AI-1), SERPINF1, SHBG, SLA2, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPP1, SPRR1B (Sprl), ST6GAL1, STAB1, STATE, STEAP, STEAP2, TIOI, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tendinogen, TRAIL receptor, TNF-α, Tn-antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, TB4R2, TBX21, TCP10 TDGF1, TEK, TGFA, TGFB1, TGFBlil, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, THBS1 (thromboretin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor, TLR10, TLR2, TLR3, TLR4, TLRS, TLR6, TLR7, TLRS, TLR9, TNF, TNF-α, TNFAIP2 (B94), TNFAIP3, TNFRSF1 1A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSFS, TNFRSF6 (Fas), TNFRSF7, TNFRSFS, TNFRSF9, TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF1S (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSFS (CD40 ligand),TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF5 (CD30 ligand), TNFSF9 (4-lBB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TPS3, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAPS, TRAF6, TREM1, TREM2, TRPC6, TSLP, TWEAK, VEGFR, ED-B fibronectin, WT-1, 17-IA antigen, complement factors C3, C3a, C3b, C5a, CS Angiogenesis markers, bcl-2, bcl-6, Kras, cMET, CD19 / CD3, BCMA / CD3, EGFR, HER3, IL17RA / IL7R, IL-6 / IL-23, IL1 / IL-8, IL-6, IL-6R / IL-21, IL-21R, ANG2 / VEGF, VEGF / PDGFR-β, vascular endothelial growth factor (VEGF) receptor 2 / CD3, PSMA / CD3, EPCAM / CD3, VEGFR-1, VEGFR-2, VEGFR-3, VEGFB, VEGFC, multifunctional proteoglycans, VHL CS, VLA-4, c-FMS / CSFIR, RET, HER3, HER4, IGFR, PDGFR, c-KIT, BCR, integrins, MMPs, VEGF, EGF, PIGF, PDGF, HGF, angiopoietin, ERBB-3 / C-MET, ERBB-2 / C-MET, EGF receptor 1 / CD3, EGFR / HER3, PSCA / CD3, C-MET / CD3, ENDOSIALIN / CD3, EPCAM / CD3, IGF-1R / CD3, FAPALPHA / CD3, EGFR / IGF-IR, IL 25 17A / F, EGF receptor 1 / CD3 and CD19 / CD16, KHI, Tn-antigen, TF-antigen, CD44, glycolipids, glycosphingolipids, such as 30 Gg3, Gb3, GD3, GD2, Gb5, Gm1, Gm2, sialytetrasaccharide ceramide, XCL1 (lymphocyte chemokine), XCL2 (SCM-1b), XCR1 (GPRS / CCXCR1), YY1, and ZFPM2.

[0736] 26. The antigen-binding molecule according to any one of Examples 1 to 24, wherein the antigen-binding molecule is capable of binding target antigen pairs selected from the group consisting of: CD137 and CD20, CD137 and EGFR, CD137 and Her-2, CD137 and PD-1, CD137 and PDL-1, VEGF and PD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1, TIM-3 and PDL-1, EGFR and DLL-4, CD138 and CD20, CDI 38 and CD40, CDI 9 and CD20, CD20 and CD3, CD3 and CD33, CD3 and CD133, CD47 and CD20, CD38 and CD138, CD38 and CD20, CD20 and CD22, CD38 and CD40, CD40 and CD20, CD-8 and IL-6, CSPGs and RGM A. CTLA-4 and BTN02, IGF1 and IGF2, IGF1 / 2 and Erb2B, IGF-1R and EGFR, EGFR and CD13, IGF-1R and ErbB3, EGFR-2 and IGFR, VEGFR-2 and Met, VEGF-A and angiopoietin-2 (Ang-2), IL-12 and TWEAK, IL-13 and IL-1β, PDGFR and VEGF, EpCAM and CD3, Her2 and CD3, CD19 and CD3, EGFR and Her3, CD16a and CD30, CD30 and PSMA, EGFR and CD3, CEA and CD3, TROP-2 and HSG, TROP-2 and CD3, MAG and RGM A, NgR and RGM A, NogoA and RGM A, OMGp and RGM A. PDL-1 and CTLA-4, CTLA-4 and PD-1, PD-1 and TIM-3, RGMA and RGM B. Te38 and TNFa, TNFa and Blys, TNFa and CD-22, TNFa and CTLA-4 domains, TNFa and GP130, TNFa and IL-12p40, and TNFa and RANK ligand.

[0737] 27. The antigen-binding molecule according to any one of Examples 1 to 24, wherein the antigen-binding molecule is capable of binding one or two cytokines, cytokine-associated proteins, and cytokine receptors selected from the group consisting of: BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFNG, IFNW1, FILI, FILI (EP SILON), FILI (ZETA), ILIA, ILIB, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, ILIO, ILi1, ILI2A, ILI2B, ILI3, ILI4, ILI5, ILI6, ILI7, ILI7B, ILI8, ILI9, IL 20. IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, FGER1, FGFR2, FGFR3, EGFR, RORI, 2B4, KIR, CD137, CD27, OX40, CD40L, A2aR, CD48, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CD70, CD40, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a), TNFSF4 (OX40 ligand) TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD), VEGF, VEGFB, VEGFC, ILIR1, ILIR2, ILIR1I, ILIRL2, IL2RA, IL2RBIL2RG, IL3RA, IL4R, IL5RA, IL6R, IL 7R, IL8RA, IL8RB, IL9R, ILIORA, ILIORB, IL11RA, ILI2RB1, ILI2RB2, ILI3RA1, ILI3RA2, ILI5RA, ILI7R, ILI8R1, IL20RA, IL2 1R, IL22R, IL1HY1, ILIRAP, ILIRAPLI, ILIRAPPL2, ILIRN, IL6S T, ILI8BP, ILI8RAP, IL22RA2, AIF1, HGF, LEP (protein), PTN, THPO.

[0738] 28. The antigen-binding molecule according to any one of Examples 1 to 24, wherein the antigen-binding molecule is capable of binding one or more chemokines, chemokine receptors, and chemokine-associated proteins selected from the group consisting of: CCLI (I-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCLII (chemokine), CCLI3 (MCP-4), CCLI5 (MIP-1d), CCLI6 (HCC-4), CCLI7 (TARC), CCLI 8 (PARC), CCLI9 (MIP-3b), CCL20 (MIP-3a), CCL21 (SLC / exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / chemokine-2), CCL25 (TECK), CCL26 (chemokine-3), CCL27 (CTACK / ILC), CCL28, CXCLI (GRO1), CXCL2 (GR02), CXCL3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCLIO (IP)10), CXCL11 (I-TAC), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1 (lymphocyte chemokine), XCL2 (SCM-1b), BLR1 (MDR15), CCBP2 (D6 / JAB61), CCR1 (CKR1 / HM145), CCR2 ( mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6 ), CCR7 (CKR7 / EBI1), CCRS (CMKBR8 / TER1 / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR 1 (GPR5 / CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSGSO), C XCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), HM74, ILSRA (IL8Ra), ILSRB (IL8Rb), LTB4R (GPR1 6), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSFS, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HIF1A, ILS, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.

[0739] 29. The antigen-binding molecule according to any one of Examples 1 to 24, wherein the antigen-binding molecule is capable of binding cytokine pairs.

[0740] 30. The antigen-binding molecule according to Example 29, wherein the bispecific antigen-binding molecule is capable of binding to cytokine pairs selected from the group consisting of: TSLP, IL-1α and IL-1β, IL-12 and IL-18, TNFα and IL-23, TNFα and IL-13, TNF and IL-18, TNF and IL-12, TNF and IL-1β, TNF and MIF, TNF and IL-6, TNF and IL-6 receptor, TNF and IL-17, IL-17 and IL-20, IL-17 and IL-18. -23, TNF and IL-15, TNF and VEGF, VEGFR and EGFR, PDGFR and VEGF, IL-13 and IL-9, IL-13 and IL-4, IL-13 and IL-5, IL-13 and IL-25, IL-13 and TARC, IL-13 and MDC, IL-13 and MIF, IL-13 and TGF-β, IL-13 and LHR agonists, IL-13 and CL25, IL-13 and SPRR2a, IL-13 and SPRR2b, IL-13 and ADAM8, as well as TNFa and PGE4, IL-13 and PED2, and TNF and PEG2.

[0741] 31. The antigen-binding molecule according to any one of Examples 1 to 30, wherein the antigen-binding molecule is capable of binding each epitope with similar or greater affinity to the bispecific antigen-binding molecule, relative to a monospecific antibody or antibody fragment specific to each epitope.

[0742] 32. The antigen-binding molecule according to any one of Examples 1 to 31, wherein the antigen-binding molecule has an agonist function.

[0743] 33. The antigen-binding molecule according to any one of Examples 1 to 31, wherein the antigen-binding molecule has a blocking function and has a similar or lower IC50 relative to the parent antibody, optionally wherein the parent antibody is a human antibody of the IgG isotype.

[0744] 34. The antigen-binding molecule according to any one of Examples 1 to 33, wherein the antigen-binding molecule is bispecific to a single ligand and forms a higher level of 1:1 ligand complex relative to one or more parental antibodies.

[0745] 35. An antigen-binding molecule comprising:

[0746] The first antigen-binding Fab domain specifically binds to the first epitope.

[0747] Specifically binds to a second antigen-binding Fab domain of a second epitope that is different from the first epitope;

[0748] Including the Fc domain of the first heavy chain polypeptide and the second heavy chain polypeptide;

[0749] A first linker is formed by connecting the N-terminus of the heavy chain of the first antigen-binding Fab domain to the C-terminus of the first heavy chain polypeptide; and

[0750] A second linker is formed by attaching the N-terminus of the heavy chain of the second antigen-binding Fab domain to the C-terminus of the second heavy chain polypeptide.

[0751] 36. The antigen-binding molecule according to Example 35, wherein the first heavy chain polypeptide comprises:

[0752] First CH amino acid sequence;

[0753] The first VH amino acid sequence; and

[0754] The second CH amino acid sequence, wherein the first VH amino acid sequence is located between the first CH amino acid sequence and the second CH amino acid sequence.

[0755] 37. The antigen-binding molecule according to Example 36, wherein the first CH amino acid sequence includes a first CH3 amino acid sequence located at the N-terminus of the first VH amino acid sequence.

[0756] 38. The antigen-binding molecule according to Example 36, wherein the first CH3 comprises an H435R mutation and a Y436F mutation.

[0757] 39. The antigen-binding molecule according to any one of Examples 35 to 38, wherein the second heavy chain polypeptide comprises:

[0758] The third CH amino acid sequence;

[0759] The second VH amino acid sequence; and

[0760] The fourth CH amino acid sequence, wherein the second VH amino acid sequence is located between the third CH amino acid sequence and the fourth CH amino acid sequence.

[0761] 40. The antigen-binding molecule according to Example 39, wherein the first CH amino acid sequence includes a first CH3 amino acid sequence located at the N-terminus of the first VH amino acid sequence.

[0762] 41. The antigen-binding molecule according to Example 40, wherein the first CH3 comprises an H435R mutation and a Y436F mutation.

[0763] 42. The antigen-binding molecule according to Example 40 or Example 41, further comprising a first adapter connecting the N-terminus of the first VH amino acid sequence to the C-terminus of the first CH3 amino acid sequence.

[0764] 43. The antigen-binding molecule according to any one of Examples 35 to 42, wherein the third CH amino acid sequence comprises a second CH3 amino acid sequence located at the N-terminus of the second VH amino acid sequence.

[0765] 44. The antigen-binding molecule according to Example 43, further comprising a second adapter connecting the N-terminus of the second VH amino acid sequence to the C-terminus of the second CH3 amino acid sequence.

[0766] 45. The antigen-binding molecule according to any one of Examples 35 to 44, wherein the second CH amino acid sequence comprises a first CH1 amino acid sequence located at the C-terminus of the first VH amino acid sequence.

[0767] 46. ​​The antigen-binding molecule according to any one of Examples 35 to 45, wherein the fourth CH amino acid sequence comprises a second CH1 amino acid sequence located at the C-terminus of the second VH amino acid sequence.

[0768] 47. The antigen-binding molecule according to any one of Examples 35 to 46, further comprising a first CH2 amino acid sequence located at the N-terminus of the first CH3 amino acid sequence.

[0769] 48. The antigen-binding molecule according to any one of Examples 35 to 47, further comprising a second CH2 amino acid sequence located at the N-terminus of the second CH3 amino acid sequence.

[0770] 49. The antigen-binding molecule according to any one of Examples 35 to 48, wherein the antigen-binding molecule does not contain hinge region disulfide bonds.

[0771] 50. The antigen-binding molecule according to any one of Examples 35 to 49, further comprising a first light chain polypeptide, the first light chain polypeptide comprising: a first VL amino acid sequence; and a first CL amino acid sequence.

[0772] 51. The antigen-binding molecule according to Example 50, further comprising a disulfide bond connecting the first CL to the first CH1.

[0773] 52. The antigen-binding molecule according to any one of Examples 35 to 51, further comprising a second light chain polypeptide, the second light chain polypeptide comprising: a second VL amino acid sequence; and a second CL amino acid sequence.

[0774] 53. The antigen-binding molecule according to Example 521, further comprising a disulfide bond connecting the second CL to the second CH1.

[0775] 54. The antigen-binding molecule according to any one of Examples 44 to 53, wherein the first adapter and the second adapter each comprise a polypeptide.

[0776] 55. The antigen-binding molecule according to Example 54, wherein the first linker and the second linker have a length of 0 to 50 amino acids.

[0777] 56. The antigen-binding molecule according to any one of Examples 54 to 55, wherein the first adapter and the second adapter have the same amino acid sequence.

[0778] 57. The antigen-binding molecule according to any one of Examples 54 to 56, wherein the first adapter and the second adapter each comprise a polyglycine and a serine amino acid sequence.

[0779] 58. The antigen-binding molecule according to Example 57, wherein the polyglycine and serine amino acid sequences comprise 2 to 6 repeating GGGGS (SEQ ID NO: 3) amino acid sequences.

[0780] 59. The antigen-binding molecule according to Example 58, wherein the polyglycine and serine amino acid sequences comprise (G4S)2 (SEQ ID NO: 18), (G4S)3 (SEQ ID NO: 4), or (G4S)4 (SEQ ID NO: 19).

[0781] 60. The antigen-binding molecule according to any one of Examples 52 to 59, wherein the first light chain polypeptide and the second light chain polypeptide have the same amino acid sequence.

[0782] 61. The antigen-binding molecule according to any one of Examples 35 to 60, wherein the first heavy chain polypeptide and the second heavy chain polypeptide have the same amino acid sequence.

[0783] 62. The antigen-binding molecule according to any one of Examples 35 to 60, wherein the first heavy chain polypeptide and the second heavy chain polypeptide have different amino acid sequences.

[0784] 63. The antigen-binding molecule according to any one of Examples 35 to 61, wherein the antigen-binding molecule is capable of binding one or more antigens selected from the group consisting of: ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVR1I, ADORA2A, Aggrecan, AGR2, AICDA, AIF1, AIG1, AKAP1, AKAP2, AMH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, AZGP1 (zinc-α-glycoprotein), ART-4, B7, B7.1, B7.2, BAD, BAFF , BAGI, BAIi, BCL2, BCL6, BDNF, BLNK, BLRl (MDRlS), BlyS, BMPl, BMP2, BMP3B (GDF10), BMP4, BMP6, BMPS, BMPR1A, BMPR1B, BMPR2, BPAG1 (lectin), BRCA1, Ba -733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-I, CASP-8 / m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CDS, CD8, CDI-IA, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD8 0. CD83, CD95, CD126, CD133, CD138, CD147, CD154, CDC27, CDK-4 / m, CDKN2A, CXCR4, CXCR7, CXCL12, C19orf10 (IL27w), C3, C4A, CS, CSR1, CANT1, CASPI, CASP4, CAV1, CCBP2 (D6 / JAB61), CCLI (I-309), CCLII (chemokine), CCL13 (MCP-4), CCLIS (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCLIS (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MIP-2), SLC, exodus-2, CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / chemokine-2), CCL2S (TECK)CCL26 (chemokine-3), CCL27 (CTACK / ILC), CCL2S, CCL3 (MIP1a), CCL4 (MIP-1b), CCLS (RANTES), CCL7 (MCP-3), CCLS (mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1 / HM14S), CCR2 (mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCRS (CMKBRSIChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EB1), CCRS (CMKBRS / TER1 / CKR-LI), CCR9 (GPR-9-6), CCRLI (VSHK1) CCRL2 (L-CCR), CD164, CDlC, CD200, CD-22, CD24, CD2S, CD3S, CD3E, CD3G, CD3Z, CD4, CD44, C D4SRB, CD47, CD4S, CDS2, CD69, CD72, CD79A, CD79B, CDSO, CDS1, CDS3, CDS6, CD137, CD13S, B7 -1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CDH1 (E-cadherin), CDH10, CDH12, CDH13, CDHlS, CDH19, CDH20, CDHS, CDH7, CDHS, CDH9, CDK2, CDK3, CDK4, CDKS, CDK6, CDK7, CDK9, CDKN1A (p21 Wap1 / Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEBPB, CER1, CHGA, CHGB, chitinase, CHST1O, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSFS, CKLFSF6, CKLFSF7, CKLFSFS, CLDN3, CLDN7 (blocking-7), CLN3, CLU (polybutase) Cluster proteins), CMKLR1, CMKOR1 (RDC1), CNR1, COLISA1, COLIA1, COL4A3, COL6A1, CR2, CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTLA-4, CTNNB1 (β-catenin), CTSB (cathepsin B), CX3CLI (SCYD1), CX3CR1 (V2S), CXCLI (GRO1), CXCLIO (IP-10)CXCL11 (I-TAC / IP-9), CXCL13, CXCL14, CXCL16, CXCL2 (GR02), CXCL3 (GR03), CXCLS (ENA-7S / LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), CYBS, CYC1, CYSLTR1, HIF-1-a, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-met, DAB2IP, DES, DKFZp4S1J01 1S, DNCLI, DPP4, DAM, EGFR, EGFRvllll, EGP-1, EGP-2, ELF2-M, Ep-CAM, E2F1, ECGF1, EDG1, EFNA1, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, EN01, EN02, E N03, EPHB4, EPO, EREG, ERKS, ESR1, ESR2, F3 (TF), FADD, FasL, FASN, FCER1A, FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF1S, FGF19, FGF2 (bFGF), FGF20, FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGFS, FGF7 (KGF), FGFS, FGF9, FGFR3, FIGF (VEGFD), FILI (EPSILON), FILI (ZETA), FLJ12SS4, FLJ2SS30, FLRT1 (fibronectin), FOS, FOSLI (FRA-1), FY (DARC), Flt-I, Flt-3, folate receptor, G250 antigen, GAGE, GROB GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GDFS, GFil, GGT1, GM-CSF, GNAS1, GNRH1, GPR2 (CCR10), GPR31, GPR44, GPRS1 (FKSGSO), GRCC10 (C10), GRP, GSN (gelatin), GSTP1, HAVCR2, HDAC4, HDACS, HDAC7A, HDAC9, HGF, HIP1 histamine and histamine receptor, HLA-A, HLA-DRA, HM74, HMOX1, HUMCYT2A, HLA-DR, HMI 24, human chorionic gonadotropin (HCG) and its subunits,HER2 / neu, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2 or 1a, IGF-IR, IFN-ɣ, IFN-α, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IGBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-1, IL-10, IL-10RA, IL-10RB, IL-11, IL-11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL-13RA1, IL-13RA2, IL-14, IL-1S, IL-1SRA, IL-16, IL-17, IL-17B, IL-17C, IL-17R, IL-18, IL-18BP, IL-18R1, IL-18RAP, IL-19, IL-IA, IL-1B, IL-1F10, IL-1FS, IL-1F6, IL-1F7, IL-1F8, IL-1F9, IL-1HY1, IL-1R1, IL-1R2, IL-1RAP, IL-1RAPL1, IL-1RAPL2, IL-1RL1, IL-1RL2 IL-1RN, IL-2, IL-20, IL-20RA, IL-21R, IL-22, IL-22R, IL-22RA2, IL-23, IL-24, IL-2S, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL-2RA, IL-2RB, IL-2RG, IL-3, IL-30, IL-3RA, IL-4, IL-4R, IL-S, IL-5RA, IL-6, IL-6R, IL-6ST (glycoprotein 130), IL-7, IL-7R, IL-S, IL-SRA, IL-SRB, IL-9, IL-9R, IL-K, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1, ITGA2, ITGA3, ITGA6 (a6 antigen), ITGAV, ITGB3, ITGB4 (b4 integrin) insulin-like growth factor-I (IGF-1), ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4IFNAS, IFNA6, IFNA7, IFNB1, IFNW1, JAG1, JAK1, JAK3, JUN, K6HF, KAi1, KDR, KITLG, KLFS (GC box BP), KLF6, KLK10, KLK12, KLK13KLK14, KLK1S, KLK3, KLK4, KLKS, KLK6, KLK9, KRT1, KRT19 (keratin 19), KRT2A, KRTHB6 (hair-specific type II keratin), KC4-antigen, KS-1-antigen, KS 1-4, Le-Y, LDR / FUT, LAMAS, LEP (leptin), Lingo-p7S, Lingo-Troy, LPS, LTA (TNF-β), LTB, LTB4R (GPR16), LTB4R2, LTBR, ​​MACMARCKS, MAG or Omgp, MAP2K7 (c-Jun), MDK, MIB1, intermediate factor, MIF, MIP-2, MKI67 (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT3 (metallothionein-III), MTSS1, MUC1 (mucin), MYC, M YD88, Macrophage Migration Inhibitor Factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, MUM-1 / 2, MUM-3, NCA66, NCA95, NCA90, NCK2, Neuroenergies, NFKB1, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgR-Nogo66 (Noga), NgRp7S, NgR- Troy, NME1 (NM23A), NOXS, NPPB, NROB1, NROB2, NR1D1, NR1D2, NRIH2, NRIH3, NRIH4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NRSA1, NRSA2, NR6A1, NRP1, NRP2, NTSE, NTN4, ODZ1, OPRD1, PCSK9, P2RX7, PAP, PART1 PATE, PAWR, PCA3, PCNA, PD-1, PD-L1, α4β7, OX40, GITR, TIM-3, Lag-3, B7-H3, B7-H4, GDFS, CGRP, Lingo-I, Factor IXa, Factor X, ICOS, GARP, BTLA, CD160, RORI, 2B4, KIR, CD27, OX40, A2aR, PDGFA, PDGFB, PECAM1, PF4 (CXCL4), PGF, PGR, phosphatidylcholine, PIAS2, PIK3CG, PLAU (uPA), PLG, PLXDC1PPBP (CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2 (COX-2), PTN, pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, 10 PIGF, ILGF, ILGF-IR, IL-6, RS5, RANTES, RAC2 (p21Rac2), RARB, RGS1, RGS13, RGS3, RNF110 (ZNF144), ROB02, S100A2, SCGB1D2 (lipophilic B), SCGB2A1 (mammary globin 2), SCGB2A2 (mammary globin 1), SCYE1 (endothelial monocyte activation cytokine), SDF2, SERPINA1, SERPINA3, SERPINABS (mammary filament inhibitory protein), SERPINE1 (P AI-1), SERPINF1, SHBG, SLA2, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPP1, SPRR1B (Sprl), ST6GAL1, STAB1, STATE, STEAP, STEAP2, TIOI, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tendinogen, TRAIL receptor, TNF-α, Tn-antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, TB4R2, TBX21, TCP10 TDGF1, TEK, TGFA, TGFB1, TGFBlil, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, THBS1 (thromboretin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIMP3, tissue factor, TLR10, TLR2, TLR3, TLR4, TLRS, TLR6, TLR7, TLRS, TLR9, TNF, TNF-α, TNFAIP2 (B94), TNFAIP3, TNFRSF1 1A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSFS, TNFRSF6 (Fas), TNFRSF7, TNFRSFS, TNFRSF9, TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF1S (VEGI), TNFSF18, TNFSF4 (OX40 ligand), TNFSFS (CD40 ligand),TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF5 (CD30 ligand), TNFSF9 (4-lBB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TPS3, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAPS, TRAF6, TREM1, TREM2, TRPC6, TSLP, TWEAK, VEGFR, ED-B fibronectin, WT-1, 17-IA antigen, complement factors C3, C3a, C3b, C5a, CS Angiogenesis markers, bcl-2, bcl-6, Kras, cMET, CD19 / CD3, BCMA / CD3, EGFR, HER3, IL17RA / IL7R, IL-6 / IL-23, IL1 / IL-8, IL-6, IL-6R / IL-21, IL-21R, ANG2 / VEGF, VEGF / PDGFR-β, vascular endothelial growth factor (VEGF) receptor 2 / CD3, PSMA / CD3, EPCAM / CD3, VEGFR-1, VEGFR-2, VEGFR-3, VEGFB, VEGFC, multifunctional proteoglycans, VHL CS, VLA-4, c-FMS / CSFIR, RET, HER3, HER4, IGFR, PDGFR, c-KIT, BCR, integrins, MMPs, VEGF, EGF, PIGF, PDGF, HGF, angiopoietin, ERBB-3 / C-MET, ERBB-2 / C-MET, EGF receptor 1 / CD3, EGFR / HER3, PSCA / CD3, C-MET / CD3, ENDOSIALIN / CD3, EPCAM / CD3, IGF-1R / CD3, FAPALPHA / CD3, EGFR / IGF-IR, IL 25 17A / F, EGF receptor 1 / CD3 and CD19 / CD16, KHI, Tn-antigen, TF-antigen, CD44, glycolipids, glycosphingolipids, such as 30 Gg3, Gb3, GD3, GD2, Gb5, Gm1, Gm2, sialytetrasaccharide ceramide, XCL1 (lymphocyte chemokine), XCL2 (SCM-1b), XCR1 (GPRS / CCXCR1), YY1, and ZFPM2.

[0785] 64. An antigen-binding molecule according to any one of Examples 35 to 61, wherein the antigen-binding molecule is capable of binding target antigen pairs selected from the group consisting of: CD137 and CD20, CD137 and EGFR, CD137 and Her-2, CD137 and PD-1, CD137 and PDL-1, VEGF and PD-L1, Lag-3 and TIM-3, OX40 and PD-1, TIM-3 and PD-1, TIM-3 and PDL-1, EGFR and DLL-4, CD138 and CD20, CDI 38 and CD40, CDI 9 and CD20, CD20 and CD3, CD3 and CD33, CD3 and CD133, CD47 and CD20, CD38 and CD138, CD38 and CD20, CD20 and CD22, CD38 and CD40, CD40 and CD20, CD-8 and IL-6, CSPGs and RGM A. CTLA-4 and BTN02, IGF1 and IGF2, IGF1 / 2 and Erb2B, IGF-1R and EGFR, EGFR and CD13, IGF-1R and ErbB3, EGFR-2 and IGFR, VEGFR-2 and Met, VEGF-A and angiopoietin-2 (Ang-2), IL-12 and TWEAK, IL-13 and IL-1β, PDGFR and VEGF, EpCAM and CD3, Her2 and CD3, CD19 and CD3, EGFR and Her3, CD16a and CD30, CD30 and PSMA, EGFR and CD3, CEA and CD3, TROP-2 and HSG, TROP-2 and CD3, MAG and RGM A, NgR and RGM A, NogoA and RGM A, OMGp and RGM A. PDL-1 and CTLA-4, CTLA-4 and PD-1, PD-1 and TIM-3, RGMA and RGM B. Te38 and TNFa, TNFa and Blys, TNFa and CD-22, TNFa and CTLA-4 domains, TNFa and GP130, TNFa and IL-12p40, and TNFa and RANK ligand.

[0786] 65. The antigen-binding molecule according to any one of Examples 35 to 61, wherein the antigen-binding molecule is capable of binding one or two cytokines, cytokine-associated proteins, and cytokine receptors selected from the group consisting of: BMP1, BMP2, BMP3B (GDF10), BMP4, BMP6, BMP8, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, and FGF12. , FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFNG, IFNW1, FILI, FILI (EP SILON), FILI (ZETA), ILIA, ILIB, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, ILIO, ILi1, ILI2A, ILI2B, ILI3, ILI4, ILI5, ILI6, ILI7, ILI7B, ILI8, ILI9, IL 20. IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, FGER1, FGFR2, FGFR3, EGFR, RORI, 2B4, KIR, CD137, CD27, OX40, CD40L, A2aR, CD48, B7-1, B7-2, ICOSL, B7-H3, B7-H4, CD137L, OX40L, CD70, CD40, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a), TNFSF4 (OX40 ligand) TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, FIGF (VEGFD), VEGF, VEGFB, VEGFC, ILIR1, ILIR2, ILIR1I, ILIRL2, IL2RA, IL2RBIL2RG, IL3RA, IL4R, IL5RA, IL6R, IL 7R, IL8RA, IL8RB, IL9R, ILIORA, ILIORB, IL11RA, ILI2RB1, ILI2RB2, ILI3RA1, ILI3RA2, ILI5RA, ILI7R, ILI8R1, IL20RA, IL2 1R, IL22R, IL1HY1, ILIRAP, ILIRAPLI, ILIRAPPL2, ILIRN, IL6S T, ILI8BP, ILI8RAP, IL22RA2, AIF1, HGF, LEP (protein), PTN, THPO.

[0787] 66. The antigen-binding molecule according to any one of Examples 35 to 61, wherein the antigen-binding molecule is capable of binding one or more chemokines, chemokine receptors, and chemokine-associated proteins selected from the group consisting of: CCLI (I-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCLII (chemokine), CCLI3 (MCP-4), CCLI5 (MIP-1d), CCLI6 (HCC-4), CCLI7 (TARC), CCLI 8 (PARC), CCLI9 (MIP-3b), CCL20 (MIP-3a), CCL21 (SLC / exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / chemokine-2), CCL25 (TECK), CCL26 (chemokine-3), CCL27 (CTACK / ILC), CCL28, CXCLI (GRO1), CXCL2 (GR02), CXCL3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCLIO (IP)10), CXCL11 (I-TAC), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP (CXCL7), CX3CL1 (SCYD1), SCYE1, XCL1 (lymphocyte chemokine), XCL2 (SCM-1b), BLR1 (MDR15), CCBP2 (D6 / JAB61), CCR1 (CKR1 / HM145), CCR2 ( mcp-1RB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6 ), CCR7 (CKR7 / EBI1), CCRS (CMKBR8 / TER1 / CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR 1 (GPR5 / CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GPR2 (CCR10), GPR31, GPR81 (FKSGSO), C XCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), HM74, ILSRA (IL8Ra), ILSRB (IL8Rb), LTB4R (GPR1 6), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSFS, BDNF, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HIF1A, ILS, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.

[0788] 67. The antigen-binding molecule according to any one of Examples 35 to 61, wherein the antigen-binding molecule is capable of binding cytokine pairs.

[0789] 68. The antigen-binding molecule according to Example 67, wherein the antigen-binding molecule is capable of binding to cytokine pairs selected from the group consisting of: TSLP, IL-1α and IL-1β, IL-12 and IL-18, TNFα and IL-23, TNFα and IL-13, TNF and IL-18, TNF and IL-12, TNF and IL-1β, TNF and MIF, TNF and IL-6, TNF and IL-6 receptor, TNF and IL-17, IL-17 and IL-20, IL-17 and IL-2 3. TNF and IL-15, TNF and VEGF, VEGFR and EGFR, PDGFR and VEGF, IL-13 and IL-9, IL-13 and IL-4, IL-13 and IL-5, IL-13 and IL-25, IL-13 and TARC, IL-13 and MDC, IL-13 and MIF, IL-13 and TGF-β, IL-13 and LHR agonists, IL-13 and CL25, IL-13 and SPRR2a, IL-13 and SPRR2b, IL-13 and ADAM8, as well as TNFa and PGE4, IL-13 and PED2, and TNF and PEG2.

[0790] 69. The antigen-binding molecule according to any one of Examples 35 to 68, wherein the antigen-binding molecule is capable of binding each epitope with similar or greater affinity to a monospecific antibody or antibody fragment specific to each epitope.

[0791] 70. The antigen-binding molecule according to any one of Examples 35 to 69, wherein the antigen-binding molecule has agonist function.

[0792] 71. The antigen-binding molecule according to any one of Examples 35 to 70, wherein the antigen-binding molecule has a blocking function and has a similar or lower IC50 relative to the parent antibody, optionally wherein the parent antibody is a human antibody of the IgG isotype.

[0793] 72. The antigen-binding molecule according to any one of Examples 35 to 71, wherein the antigen-binding molecule is bispecific to a single ligand and forms a higher level of 1:1 ligand complex relative to one or more parental antibodies.

[0794] 73. The antigen-binding molecule according to any one of Examples 35 to 72, wherein the antigen-binding molecule is conjugated with an agent selected from the group consisting of: immunoadhesin molecules, imaging agents, therapeutic agents, and cytotoxic agents.

[0795] 74. A pharmaceutical composition comprising an antigen binder according to any one of Examples 1 to 73, and a pharmaceutically acceptable carrier.

[0796] 75. A nucleic acid molecule encoding an antigen-binding molecule according to any one of Examples 1 to 73.

[0797] 76. The nucleic acid molecule according to Example 74, wherein the nucleic acid molecule is operatively linked to an expression control sequence.

[0798] 77. An expression vector comprising a nucleic acid molecule as described in Example 75 or 76.

[0799] 78. A host cell comprising a nucleic acid molecule as described in Example 75 or 76 or a vector as described in Example 77.

[0800] 79. The host cell according to Example 78, wherein the cell is a eukaryotic cell.

[0801] 80. The host cell according to Example 78 or 79, wherein the cell is an animal cell.

[0802] 81. The host cell according to any one of Examples 78 to 79, wherein the cell is a mammalian cell, optionally a CHO cell.

[0803] 82. A method of treating a subject suffering from symptoms related to any one or more antigens described in Example 63, the method comprising administering to the subject an effective amount of an antigen-binding molecule according to any one of Examples 1 to 73.

[0804] 83. A method for inhibiting a molecular pathway in a subject, the method comprising administering to the subject an effective amount of an antigen-binding molecule according to any one of Examples 1 to 73 to inhibit the molecular pathway.

[0805] 84. A method for activating a molecular pathway in a subject, the method comprising administering to the subject an effective amount of an antigen-binding molecule according to any one of Examples 1 to 73 to activate the molecular pathway.

[0806] 85. Use of an antigen-binding molecule according to any one of Examples 1 to 73 in the preparation of a medicament for treating symptoms associated with any one or more antigens described in Example 63.

[0807] This application also relates to the following implementation schemes:

[0808] 1. An antigen-binding molecule that binds to a first target molecule and comprises:

[0809] (a) A first polypeptide, wherein the first polypeptide comprises, in the N-to-C-terminal orientation:

[0810] (i) The first Fc structural domain; and

[0811] (ii) a first Fab domain, the first Fab domain comprising a first heavy chain variable region (VH) associated with a first light chain variable region (VL); and

[0812] (b) A second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation:

[0813] (i) The second Fc structural domain; and

[0814] (ii) A second Fab domain, the second Fab domain comprising a second VH associated with a second VL,

[0815] The first Fc structural domain and the second Fc structural domain associate with each other to form the Fc region.

[0816] 2. The antigen-binding molecule according to claim 1, comprising a first linker between the first Fc domain and the first VH.

[0817] 3. The antigen-binding molecule according to claim 2, wherein the first linker is 5 to 60 amino acids in length, 10 to 60 amino acid residues in length, 5 to 20 amino acid residues in length, 5 to 30 amino acid residues in length, 10 to 30 amino acid residues in length, 10 to 20 amino acid residues in length, 20 to 50 amino acids in length, or 25 to 35 amino acids in length.

[0818] 4. The antigen-binding molecule according to claim 2 or 3, wherein the first linker comprises G n S or SG n The polymer of , optionally where n is an integer from 1 to 7.

[0819] 5. The antigen-binding molecule according to claim 4, wherein the first linker comprises a polymer of G4S (SEQ ID NO: 3), optionally wherein the first linker comprises 2 to 6 repeats of G4S (SEQ ID NO: 3), optionally wherein the first linker comprises (G4S)2 (SEQ ID NO: 18), (G4S)3 (SEQ ID NO: 4) or (G4S)4 (SEQ ID NO: 19).

[0820] 6. The antigen-binding molecule according to any one of items 2 to 5, comprising a second linker between the second Fc domain and the second VH, optionally wherein the first linker and the second linker have the same amino acid sequence.

[0821] 7. The antigen-binding molecule according to claim 6, wherein the second linker is 5 to 60 amino acids in length, 10 to 60 amino acids in length, 5 to 20 amino acid residues in length, 5 to 30 amino acid residues in length, 10 to 30 amino acid residues in length, 10 to 20 amino acid residues in length, 20 to 50 amino acids in length, or 25 to 35 amino acids in length.

[0822] 8. The antigen-binding molecule according to claim 6 or 7, wherein the second linker comprises G n S or SG n The polymer of , optionally where n is an integer from 1 to 7.

[0823] 9. The antigen-binding molecule according to claim 8, wherein the second linker comprises a polymer of G4S (SEQ ID NO: 3), optionally wherein the second linker comprises 2 to 6 repeats of G4S (SEQ ID NO: 3), optionally wherein the second linker comprises (G4S)2 (SEQ ID NO: 18), (G4S)3 (SEQ ID NO: 4) or (G4S)4 (SEQ ID NO: 19).

[0824] 10. The antigen-binding molecule according to any one of claims 1 to 9, wherein the first polypeptide includes a first hinge domain located at the N-terminus of the first Fc domain and the second polypeptide includes a second hinge domain located at the N-terminus of the second Fc domain.

[0825] 11. The antigen-binding molecule according to claim 10, wherein the first hinge domain and the second hinge domain are connected by disulfide bonds.

[0826] 12. The antigen-binding molecule according to claim 10, wherein the first hinge domain and the second hinge domain are not connected by disulfide bonds.

[0827] 13. The antigen-binding molecule according to any one of claims 1 to 12, wherein the first polypeptide does not include a VH located at the N-terminus of the first Fc domain and / or wherein the second polypeptide does not include a VH located at the N-terminus of the second Fc domain.

[0828] 14. The antigen-binding molecule according to any one of items 1 to 13, having a hinge region.

[0829] 15. The antigen-binding molecule according to item 14, which has Figure 13A or Figure 13C The hinge type shown.

[0830] 16. The antigen-binding molecule according to any one of items 1 to 13, having two hinge regions.

[0831] 17. The antigen-binding molecule according to item 16, having Figure 13B The hinge type shown.

[0832] 18. The antigen-binding molecule according to any one of items 1 to 17, wherein the first polypeptide and the second polypeptide are identical.

[0833] 19. The antigen-binding molecule according to any one of items 1 to 17, wherein the first polypeptide and the second polypeptide are not the same.

[0834] 20. The antigen-binding molecule according to any one of claims 1 to 19, wherein the first VL and the second VL are universal light chains, or wherein the light chain constant region and the first heavy chain constant region (CH1) of the first Fab domain or the second Fab domain are arranged in a crossmab configuration.

[0835] 21. The antigen-binding molecule according to any one of claims 1 to 20, wherein the first Fab domain and the second Fab domain are not in the form of a single-chain Fab.

[0836] 22. The antigen-binding molecule according to any one of claims 1 to 21, which binds the first target molecule with greater affinity and / or affinity than a natural immunoglobulin comprising the first Fab domain and the second Fab domain.

[0837] 23. The antigen-binding molecule according to any one of items 1 to 22 is divalent.

[0838] 24. The antigen-binding molecule according to any one of items 1 to 23, wherein it is an antagonist of the first target molecule.

[0839] 25. The antigen-binding molecule according to any one of claims 1 to 24, which inhibits the binding of the first target molecule to a binding partner, optionally wherein the binding partner is a receptor for the first target molecule.

[0840] 26. The antigen-binding molecule according to any one of claims 1 to 25, wherein the Fc region comprises a human Fc sequence.

[0841] 27. The antigen-binding molecule according to any one of claims 1 to 26, wherein the Fc region comprises a human IgG1 or human IgG4 Fc sequence.

[0842] 28. The antigen-binding molecule according to any one of claims 1 to 27, wherein the Fc region comprises an Fc heterodimer, optionally wherein the Fc domain in the Fc heterodimer comprises an intrapore toggle mutation compared to the wild-type Fc domain.

[0843] 29. The antigen-binding molecule of claim 28, wherein the Fc domain of the first polypeptide includes a button mutation and the Fc domain of the second polypeptide includes a pore mutation.

[0844] 30. The antigen-binding molecule according to claim 28, wherein the Fc domain of the second polypeptide includes a button mutation and the Fc domain of the first polypeptide includes a pore mutation.

[0845] 31. The antigen-binding molecule according to claim 26, wherein the Fc region comprises a star mutation compared to the wild-type Fc region.

[0846] 32. The antigen-binding molecule according to claim 26, wherein the Fc domain of the first polypeptide comprises an H435R mutation and a Y436F mutation.

[0847] 33. The antigen-binding molecule according to claim 26, wherein the Fc domain of the second polypeptide comprises an H435R mutation and a Y436F mutation.

[0848] 34. The antigen-binding molecule according to any one of claims 1 to 33, wherein the CL and CH1 in the first Fab domain are linked by disulfide bonds and / or wherein the CL and CH1 in the second Fab domain are linked by disulfide bonds.

[0849] 35. The antigen-binding molecule according to any one of claims 1 to 34, wherein the first Fab domain and the second Fab domain bind to the first target molecule.

[0850] 36. The antigen-binding molecule according to any one of claims 1 to 35, wherein the first target molecule ...

Claims

1. An antigen-binding molecule that binds to a first target molecule and comprises: (a) A first polypeptide, wherein the first polypeptide comprises, in the N-to-C-terminal orientation: (i) The first Fc structural domain; and (ii) a first Fab domain, the first Fab domain comprising a first heavy chain variable region (VH) associated with a first light chain variable region (VL); and (b) A second polypeptide, wherein the second polypeptide comprises, in the N-to-C-terminal orientation: (i) The second Fc structural domain; and (ii) A second Fab domain, the second Fab domain comprising a second VH associated with a second VL, The first Fc structural domain and the second Fc structural domain associate with each other to form the Fc region.

2. The antigen-binding molecule according to claim 1, comprising a first linker between the first Fc domain and the first VH.

3. The antigen-binding molecule according to claim 2, wherein the first linker has a length of 5 to 60 amino acids, a length of 10 to 60 amino acid residues, a length of 5 to 20 amino acid residues, a length of 5 to 30 amino acid residues, a length of 10 to 30 amino acid residues, a length of 10 to 20 amino acid residues, a length of 20 to 50 amino acids, or a length of 25 to 35 amino acids.

4. The antigen-binding molecule according to claim 2 or claim 3, wherein the first linker comprises G n S or SG n The polymer of , optionally where n is an integer from 1 to 7.

5. The antigen-binding molecule according to claim 4, wherein the first linker comprises a polymer of G4S (SEQ ID NO: 3), optionally wherein the first linker comprises 2 to 6 repeats of G4S (SEQ ID NO: 3), optionally wherein the first linker comprises (G4S)2 (SEQ ID NO: 18), (G4S)3 (SEQ ID NO: 4) or (G4S)4 (SEQ ID NO: 19).

6. The antigen-binding molecule according to any one of claims 2 to 5, comprising a second linker between the second Fc domain and the second VH, optionally wherein the first linker and the second linker have the same amino acid sequence.

7. The antigen-binding molecule according to claim 6, wherein the second linker is 5 to 60 amino acids long, 10 to 60 amino acids long, 5 to 20 amino acid residues long, 5 to 30 amino acid residues long, 10 to 30 amino acid residues long, 10 to 20 amino acid residues long, 20 to 50 amino acids long, or 25 to 35 amino acids long.

8. The antigen-binding molecule according to claim 6 or claim 7, wherein the second linker comprises G n S or SG n The polymer of , optionally where n is an integer from 1 to 7.

9. The antigen-binding molecule of claim 8, wherein the second linker comprises a polymer of G4S (SEQ ID NO: 3), optionally wherein the second linker comprises 2 to 6 repeats of G4S (SEQ ID NO: 3), optionally wherein the second linker comprises (G4S)2 (SEQ ID NO: 18), (G4S)3 (SEQ ID NO: 4) or (G4S)4 (SEQ ID NO: 19).

10. The antigen-binding molecule according to any one of claims 1 to 9, wherein the first polypeptide comprises a first hinge domain located at the N-terminus of the first Fc domain and the second polypeptide comprises a second hinge domain located at the N-terminus of the second Fc domain.

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