Engineered heavy chain variable domains and uses thereof

Abstract

The present disclosure relates to engineered heavy chain variable domains. Also disclosed are antigen binding molecules, including multivalent and multispecific antigen binding molecules, comprising such engineered heavy chain variable domains. Further disclosed are methods of producing the antigen binding molecules, pharmaceutical compositions comprising the antigen binding molecules, nucleic acids expressing the antigen binding molecules, and methods of using the antigen binding molecules.

Description

[0001] 1. Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 598,776, filed November 14, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] 2. Sequence List

[0004] This application contains a list of electronically submitted sequences, which are hereby incorporated by reference in their entirety. The copy created on November 5, 2024, is named RGN-031WO_SL.xml and is 126,612 bytes in size. Background Technology

[0005] Various biological therapeutic agents have been developed for the prevention and treatment of a variety of diseases, including proliferative diseases (e.g., cancer), chronic inflammatory diseases (e.g., Crohn's disease), and rheumatic diseases (e.g., rheumatoid arthritis).

[0006] In biotherapeutic agents, antibodies and related antigen-binding molecules have proven effective in clinical practice. However, the immunogenicity of these molecules can challenge their practicality and hinder the development of new molecules for clinical application. For example, even fully human therapeutic monoclonal antibodies can trigger the production of antidrug antibodies (ADAs), which can lead to undesirable effects such as suboptimal targeting, loss of potency, formation of highly immunogenic complexes, and other adverse events (Koren et al., 2002, Curr Pharm Biotechnol, 3(4):349-60; Schellekens, 2002, Clin Ther, 24(11):1720-40; van Schie et al., 2015, Ann Rheum Dis.74(1):311–314; Hansel, 2010, Nat Rev Drug Discov. 9(4):325–338), which may limit the clinical applicability and use of antigen-binding molecules and other biotherapeutic agents.

[0007] Therefore, there is a need in the art to mitigate the generation and / or binding of ADA associated with antigen-binding molecules and other biological products. Summary of the Invention

[0008] This disclosure relates to engineered heavy chain variable domains (VH) and antigen-binding molecules containing the engineered VH.

[0009] In particular, this disclosure provides VHs engineered to incorporate an N-glycosylation site near their C-terminus. Without being bound by theory, it is believed that the incorporation of the N-glycosylation site reduces binding to antidrug antibodies and thus reduces the antigenicity of antigen-binding molecules incorporating engineered VHs compared to unengineered VHs.

[0010] This disclosure further provides polypeptides comprising the VH of this disclosure, having an N-glycosylation site near its C-terminus. In some embodiments, the polypeptide is an antigen-binding molecule. Engineered VHs can be advantageously used in the context of a variety of antigen-binding molecules and their components, such as scFvs, Fabs, antibodies, antibody fragments, and other antigen-binding molecules, including multivalent and / or multispecific antigen-binding molecules. In addition to the engineered VHs of this disclosure, antigen-binding molecules may include one or more additional target-binding domains (e.g., one or more Fab moieties, one or more scFv moieties, or combinations thereof) and / or one or more linkers separating one or more moieties in the antigen-binding molecule.

[0011] Exemplary engineered VHs are disclosed in section 6.2. Exemplary antigen-binding molecules containing engineered VHs are disclosed in section 6.3 and in numbered Examples 1 through 106. Exemplary target-binding domains for incorporation into antigen-binding molecules are disclosed in sections 6.4.2 and 6.4.3, and exemplary connectors for connecting constant domains to target-binding domains or different components of target-binding domains are described in section 6.4.4.

[0012] This disclosure further provides nucleic acids encoding the VH and antigen-binding molecules of this disclosure. The nucleic acid encoding the antigen-binding molecule may be a single nucleic acid (e.g., a vector encoding two or more polypeptide chains) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains). This disclosure further provides host cells and cell lines engineered to express the nucleic acids, VH, and antigen-binding molecules of this disclosure. Exemplary nucleic acids, host cells, and cell lines are described in section 6.5 and numbered Examples 108 through 110.

[0013] Methods for generating antigen-binding molecules, methods for using engineered VH of this disclosure to reduce the binding of antigen-binding molecules to anti-drug antibodies, and methods for reducing the antigenicity of antigen-binding molecules are described in section 6.7 and numbered Examples 111 and 118 to 150.

[0014] This disclosure further provides pharmaceutical compositions comprising the antigen-binding molecules of this disclosure. Exemplary compositions are described in section 6.6 and in Example 107.

[0015] Methods of administering the antigen-binding molecules of this disclosure are also disclosed, including therapeutic methods comprising administering the antigen-binding molecules. Exemplary administration methods, including therapeutic methods, are described in section 6.7 and numbered Examples 112 to 114. Attached Figure Description

[0016] Figures 1A to 1C This disclosure illustrates an exemplary antigen-binding molecule comprising the amino acid sequence X1X2X3X4X5NX6X7X8X9X at its C-terminus. 10 X 11 X 12 VH of (SEQ ID NO:1), where (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, optionally wherein the amino acid is not proline; (c) X7 is S or T, and (d) X8, X9, X 10 X 11 and X 12 Each amino acid is independently selected from: any amino acid and those that are absent. This sequence is in Figures 1A to 1C The Chinese text represents it as "X1X2X3X4X5NX6[S / T]X8X9X". 10 X 11 X 12 ". Figure 1A An scFv containing VH is shown, which contains the amino acid sequence of SEQ ID NO:1 at its C-terminus. Figure 1B The diagram illustrates a bivalent antigen-binding molecule comprising two scFv molecules linked by a linker, wherein at least one of the VH molecules contains the sequence X1X2X3X4X5NX6[S / T]X8X9X at its C-terminus. 10 X 11 X 12 . Figure 1C An exemplary multispecific antigen-binding molecule comprising a first polypeptide and a second polypeptide is shown. The first polypeptide comprises, from its N-terminus to its C-terminus: a first targeting moiety (e.g., Fab or scFv), a first dimerizing moiety (e.g., an Fc domain), an optional linker, and a first scFv; the second polypeptide comprises, from its N-terminus to its C-terminus: a second targeting moiety (e.g., the Fab of the scFv), a second dimerizing moiety, an optional linker, and a second scFv, wherein each scFv contains the sequence X1X2X3X4X5NX6[S / T]X8X9X at its C-terminus. 10 X 11 X 12 VH.

[0017] Figures 2A to 2GCertain multispecific antigen-binding molecules of this disclosure are shown, each comprising a first polypeptide and a second polypeptide. The first polypeptide comprises, from the N-terminus to the C-terminus, a first targeting portion (e.g., Fab or scFv), a first dimerizing portion (e.g., an Fc domain), an optional linker, and a second targeting portion. The second polypeptide comprises, from the N-terminus to the C-terminus, a target antigen-binding portion, such as a tumor-associated antigen-targeting portion (e.g., Fab or scFv that specifically binds to a tumor-associated antigen), a second dimerizing portion, an optional linker, and a second target antigen-binding portion. Figure 2A A multispecific antigen-binding molecule is shown, whose scFv C-terminus contains the amino acid sequence VTVSS (SEQ ID NO:62). Figures 2B to 2G A multispecific antigen-binding molecule containing an engineered scFv C-terminal sequence is shown, with modifications presented in bold. Figure 2B A construct with the engineered scFv C-terminal amino acid sequence VTVSSPP (SEQ ID NO:63) is shown. Figure 2C A construct with the engineered scFv C-terminal amino acid sequence VTVKPGG (SEQ ID NO:64) is shown. Figure 2D A construct with the engineered scFv C-terminal amino acid sequence VTVKPGG (SEQ ID NO:64) and the V11K mutation is shown. Figure 2E A construct with the engineered scFv C-terminal amino acid sequence VTVSSGGGGS (SEQ ID NO:65) is shown. Figure 2F A construct with the engineered scFv C-terminal amino acid sequence VTVNSS (SEQ ID NO:50) is shown. Figure 2G A construct with the engineered scFv C-terminal amino acid sequence VTVNST (SEQ ID NO:51) is shown. Although Figures 2A to 2G The two targeting regions of each polypeptide are described as containing the same VH and VL domains, but they may contain different VH and VL domains that can recognize the same epitope, different epitopes of the same antigen, or different antigens.

[0018] Figures 3A to 3C This study demonstrates that amino acid modifications to the C-terminus of the antigen-binding molecule REGN9930 do not affect its binding affinity to target cell surface antigens expressed on Raji and Jurkat cells. Figure 3A The binding of antigen-binding molecules to RajiMAGEA4 (230-239) cells was shown. Figure 3B The binding of the antigen-binding molecule to Raji MAGEA4 (286-294) is shown. Figure 3CThe binding of antigen-binding molecules to Jurkat cells is shown.

[0019] Figure 4 This demonstrates that C-terminal amino acid modification does not affect the cytotoxic efficacy of the antigen-binding molecule REGN9930.

[0020] Figures 5A to 5E SEC-MS analysis of the antigen-binding molecule REGN9930-VNSS (C-terminal sequence VTVNSS; SEQ ID NO:50) after limited LysC digestion is shown. Figure 5A Natural SEC-UV / MS analysis is shown, with boxes labeled (i) to (iv) indicating... Figures 5B to 5E The fragment shown in the medium mass spectrometry analysis.

[0021] Figures 6A to 6D SEC-MS analysis of the antigen-binding molecule REGN9930-VNST (C-terminal sequence VTVNST; SEQ ID NO:51) after limited LysC digestion is shown. Figure 6A Natural SEC-UV / MS analysis is shown, with boxes labeled (i) through (iii) indicating... Figures 6B to 6D The fragment shown in the medium mass spectrometry analysis.

[0022] Figure 7 This indicates that, compared to REGN9930, the pre-existing ADA reactivity of antigen-binding molecules with C-terminal amino acid modifications is reduced. Detailed Implementation

[0023] 6.1. Definition

[0024] Approximately Throughout the specification, the terms “about,” “approximately,” etc., are used before numbers to indicate that the number is not necessarily precise (e.g., considering variations in fractions, measurement accuracy and / or precision, timing, etc.). It should be understood that disclosures of “about X” or “approximately X,” where X is a number, are also disclosures of “X.” Thus, for example, a disclosure of an embodiment in which one sequence has “about X% sequence identity” with another sequence is also a disclosure of an embodiment in which that sequence has “X% sequence identity” with another sequence.

[0025] and, or Unless otherwise stated, the conjunction “or” should be used in its proper sense as a Boolean logical operator, encompassing the selection of features in alternatives (A or B, where the selection of A and B are mutually exclusive) and the selection of joint features (A or B, where both A and B are selected). In some places in the text, the terms “and / or” are used for the same purpose, which should not be interpreted as implying that “or” is used to refer to mutually exclusive alternatives.

[0026] Anti-drug antibodies or ADA The term "antidrug antibody" or "ADA" refers to an antibody that specifically binds to any region of a drug. In some embodiments, a "drug" is or contains an antibody (as defined below). When a drug is or contains an antibody, an antidrug antibody can be an antibody or fragment thereof that specifically binds to a region of the drug antibody (e.g., a variable domain, constant domain, or sugar structure of the antibody). Such antidrug antibodies may appear as an immunogenic response in a patient during drug therapy. ADA can be any human immunoglobulin isotype (e.g., IgM, IgE, IgA, IgG, IgD) or IgG subclass (IgG1, 2, 3, and 4). ADA includes ADA from any animal source, including, for example, human or non-human animal (e.g., veterinary) sources.

[0027] AntibodyAs used herein, the term "antibody" refers to a polypeptide (or group of polypeptides) of the immunoglobulin family that can bind nonvalently, reversibly, and specifically to an antigen. For example, a naturally occurring IgG-type "antibody" is a tetramer consisting of at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region contains one domain (abbreviated as CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), within which more conserved regions called framework regions (FRs) are interspersed. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of both the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelified antibodies, chimeric antibodies, bispecific or multispecific antibodies, and anti-idiotype (anti-id) antibodies. Antibodies can be any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Both the light and heavy chains are divided into structural and functional homologous regions. The terms "constant" and "variable" are used functionally. In this respect, it should be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) endow important biological properties such as secretion, transplacental migration, Fc receptor binding, and complement binding. By convention, the constant domains are numbered progressively as they move further away from the antibody's antigen-binding domain or N-terminus. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains represent the C-termini of the heavy and light chains of the native antibody, respectively. For convenience, and unless the context otherwise requires, references to antibodies refer to antibody fragments and engineered antibodies that include non-naturally occurring antigen-binding domains and / or antigen-binding domains with non-natural conformations.

[0028] Antigen-binding domainAs used herein, the term "antigen-binding domain" or "ABD" refers to a portion of a binding molecule (e.g., a multispecific binding molecule, antibody, or antibody fragment) that has the ability to bind non-covalently, reversibly, and specifically to a target molecule (e.g., an antigen). Examples of antibody fragments that may contain an ABD include, but are not limited to, single-chain Fv (scFv), Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; bivalent F(ab)2 fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of VL and VH domains of an antibody single arm; dAb fragments (Ward et al., 1989, Nature 341:544-546) consisting of a VH domain; and separated complementarity-determining regions (CDRs). Thus, the term "antibody fragment" encompasses both proteolytic fragments of antibodies (e.g., Fab and F(ab)2 fragments) and engineered proteins containing one or more portions of an antibody (e.g., scFv). Antibody fragments can also be incorporated into single-domain antibodies, large antibodies, small antibodies, intracellular antibodies, biantibodies, triantibodies, tetraantibodies, v-NARs, and bi-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126-1136).

[0029] Association In the context of antigen-binding molecules, the term "association" refers to a functional relationship between two or more polypeptide chains or portions thereof. Specifically, the term "association" means that two or more polypeptides associate with each other, for example, non-covalently through molecular interactions or covalently through one or more disulfide bridges or chemical crosslinks, to produce a functional antigen-binding molecule. Examples of association that may exist in the antigen-binding molecules of this disclosure include (but are not limited to) association between homodimeric or heterodimeric Fc domains in the Fc region, association between the VH and VL regions in Fab or scFv, association between CH1 and CL in Fab, and association between CH3 and CH3 in domain-substituted Fab.

[0030] Bivalent As used herein with respect to antigen-binding molecules, the term "bivalent" refers to an antigen-binding molecule 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 of the same target molecule. In still other embodiments, the two antigen-binding sites specifically bind to different epitopes of two different target molecules. Therefore, a bivalent antigen-binding molecule can be monospecific or bispecific.

[0031] Cancer antigensThe term "cancer antigen" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed entirely or as a fragment (e.g., MHC / peptide) on the surface of cancer cells and can be used to preferentially target pharmacological agents to cancer cells. In some embodiments, cancer antigens are markers expressed by both normal cells and cancer cells, such as lineage markers, such as CD19 on B cells. In some embodiments, cancer antigens are cell surface molecules overexpressed in cancer cells compared to normal cells, such as 1-fold, 2-fold, 3-fold, or more overexpression compared to normal cells. In some embodiments, cancer antigens are cell surface molecules inappropriately synthesized in cancer cells, such as molecules containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, cancer antigens are expressed entirely or as a fragment (e.g., MHC / peptide) specifically on the cell surface of cancer cells and are not synthesized or expressed on the surface of normal cells. Therefore, the term "cancer antigen" encompasses antigens that are specific to cancer cells and are sometimes referred to in the art as tumor-specific antigens ("TSA").

[0032] Complementary Determinant Region or CDRAs used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within the variable region of an antibody that confers antigen specificity and binding affinity. Typically, three CDRs (CDR-H1, CDR-H2, CDR-H3) are present in each heavy chain variable region, and three CDRs (CDR1-L1, CDR-L2, CDR-L3) are present in each light chain variable region. Exemplary specifications that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, the ABD 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, J. Mol. Biol. 273:927-948 (Chothia numbering scheme); Martin et al., 1989, Proc. Natl. Acad. Sci. USA 86:9268-9272 (ABD numbering scheme); and Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 (IMGT numbering scheme). For example, in the classical form, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3). According to Chothia, the CDR amino acids in VH are numbered 26-32 (CDR-H1), 52-56 (CDR-H2), and 95-102 (CDR-H3); and the amino acid residues in VL are numbered 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3). By combining the CDR definitions of Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in human VH and amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in human VL.Under IMGT, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR-H1), 51-57 (CDR-H2), and 93-102 (CDR-H3), and the CDR amino acid residues in VL are numbered approximately 27-32 (CDR-L1), 50-52 (CDR-L2), and 89-97 (CDR-L3) (according to "Kabat" numbering). Under IMGT, the CDR region of the antibody can be determined using the IMGT / DomainGap Align procedure. Public databases are available for identifying CDR sequences within antibodies.

[0033] Dimerization portion The term "dimerization moiety" refers to a polypeptide chain or amino acid sequence capable of promoting association between two polypeptide chains to form a dimer. A first dimerization moiety may associate with the same second dimerization moiety, or with a second dimerization moiety different from the first dimerization moiety. In some embodiments, the dimerization moiety is an Fc domain (e.g., an Fc domain selected from isotypes IgG1, IgG2, IgG3, and IgG4, and any allotype of IgG within each isotype group), wherein two Fc domains associate to form an Fc region. Thus, the Fc region can be homodimer or heterodimer. In some embodiments, the dimerization moiety is an Fc domain or an amino acid sequence of 1 to about 200 amino acids containing at least one cysteine ​​residue. In other embodiments, the dimerization moiety is a cysteine ​​residue or a short peptide containing cysteine. Other dimerization moieties include peptides or polypeptides comprising or composed of leucine zippers, helical-cyclic motifs, or coiled-coil motifs.

[0034] EC50 The term "EC50" refers to the half-maximal effective concentration of a molecule (such as an antigen-binding molecule), which elicits half the response between baseline and maximum after a specific exposure time. EC50 essentially represents the concentration of molecules at which 50% of their maximum effect is observed. Therefore, as the EC50, or half-maximal effective concentration, increases, a decreased or weaker binding is observed.

[0035] Epitope An epitope, or antigenic determinant, is a portion of an antigen recognized by an antibody or other antigen-binding moiety as described herein. Epitopes can be linear or conformational. As described herein, an epitope can be described as "a sequence containing a specific region (e.g., a protein domain) of an antigen." Such descriptions include both linear and conformational epitopes and describe epitopes containing at least one amino acid present in a specific region of the antigen. Such epitopes may or may not contain additional amino acids not present in the specific region.

[0036] FabIn the context of the antigen-binding molecules disclosed herein, the term "Fab" refers to a pair of polypeptide chains, the first polypeptide chain containing a variable heavy (VH) domain of the antibody at the N-terminus of a first constant domain (referred to herein as C1), and the second polypeptide chain containing a variable light (VL) domain of the antibody at the N-terminus of a second constant domain (referred to herein as C2), which is capable of pairing with the first constant domain. In native antibodies, VH is located at the N-terminus of the first constant domain (CH1) of the heavy chain and VL is located at the N-terminus of the constant domain of the light chain (CL). The Fab of this disclosure may be arranged according to the native orientation or include domain substitutions or exchanges that facilitate correct VH and VL pairing. For example, the CH1 and CL domain pairs in Fab may be replaced with CH3 domain pairs to facilitate correct modified Fab chain pairing in a heterodimer molecule. CH1 and CL may also be reversed, such that CH1 is attached to VL and CL is attached to VH; this configuration is commonly referred to as a Crossmab (a "domain exchange" arrangement). Alternatively, or in addition to using substituted or exchanged constant structural domains, proper chain pairing can also be achieved by using a universal light chain capable of pairing with the two variable regions of the heterodimeric antigen-binding molecule of this disclosure. The term "Fab" encompasses single-chain Fab.

[0037] Fc 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 can be identical or different from each other. In natural antibodies, the Fc domains are typically identical, but one or both Fc domains can be advantageously modified to allow heterodimerization, for example via knock-in-hole interactions, and / or for purification, for example via star mutations.

[0038] Fv The term "Fv" refers to the smallest antibody fragment that can be derived from an immunoglobulin containing a complete target recognition and binding site. This region consists of a tightly bound, non-covalently associated dimer of a heavy chain variable domain and a light chain variable domain (VH-VL dimer). It is in this conformation that the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer antibody target binding specificity. However, in some cases, even a single variable domain (or half of an Fv containing only the three CDRs specific to the target) can have the ability to recognize and bind to the target. The mention of the VH-VL dimer in this document is not intended to convey any particular conformation. When present on a single polypeptide chain (e.g., scFv), the VH can be located at the N-terminus or C-terminus of the VL.

[0039] Half antibody: The term "half-antibody" refers to a molecule containing at least one Fc domain and capable of associating with another Fc-containing molecule through, for example, disulfide bridges or molecular interactions. A half-antibody can consist of one or more polypeptide chains (e.g., two polypeptide chains of Fab). Examples of half-antibodies are molecules containing both the heavy and light chains of an antibody (e.g., an IgG antibody). Another example of a half-antibody is a molecule containing a first polypeptide chain containing VL and CL domains and a second polypeptide chain containing VH, CH1, hinge, CH2, and CH3 domains, wherein the VL and VH domains form an ABD. Yet another example of a half-antibody is a polypeptide containing scFv, CH2, and CH3 domains. The term "half-antibody" is used for descriptive purposes only and does not imply a specific configuration or method of manufacture. Describing half-antibodies as "first" half-antibody, "second" half-antibody, "left" half-antibody, "right" half-antibody, etc., is merely for convenience and descriptive purposes.

[0040] Host cells or recombinant host cells As used herein, the terms "host cell" and "recombinant host cell" refer to cells that have been genetically engineered, for example, by introducing heterologous nucleic acids. It should be understood that such terms refer not only to specific target cells but also to the progeny of such cells. Because certain modifications may occur in offspring due to mutations or environmental influences, these progeny may actually differ from the parent cells but are still included within the scope of the term "host cell" as used herein. Host cells may, for example, transiently carry heterologous nucleic acids on extrachromosomal heterologous expression vectors, or stably carry heterologous nucleic acids, for example, by integrating them into the host cell genome. For the purpose of expressing antigen-binding molecules, host cells can be mammalian-derived or mammalian-like cell lines, such as monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, juvenile hamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NSO, PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2), SP2 / 0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphoma cells, or derivatives thereof and / or engineered variants. Engineered variants include, for example, glycan-modified and / or site-specific integration site derivatives.

[0041] Multispecific antigen-binding moleculesAs used herein, the term "multispecific antigen-binding molecule" (also referred to as "multispecific antigen-binding molecule" or "multispecific binding molecule") refers to a molecule (e.g., an assembly of multiple polypeptide chains) that comprises two half-antibodies and specifically binds to at least two different epitopes (and in some cases three, four, or more different epitopes). The multispecific antigen-binding molecules of this disclosure can be bivalent, trivalent, tetravalent, or other multivalent, and can be monospecific, bispecific, or other multispecific. The multispecific antigen-binding molecules of this disclosure can specifically bind to epitopes on one, two, three, four, or more different antigens.

[0042] Multi-price As used in this article, the term "multivalent" refers to an antigen-binding molecule containing two or more ABDs on one, two or more polypeptide chains.

[0043] N-glycosylation site As used herein, the term "N-glycosylation site" refers to an asparagine (Asn;N) residue in a glycosylated polypeptide. A classic "N-glycosylation site" contains an Asn residue within the triamino acid sequence Asn-Xaa-Ser or Asn-Xaa-Thr, where Xaa is any amino acid, typically not proline. This triamino acid sequence is generally referred to herein as "NX[S / T]". The term "N-glycosylation site" is used to describe such asparagine residues, regardless of whether an oligosaccharide is attached to that residue. A polypeptide described herein as containing an N-glycosylation site at a specific location means that the Asn residue at that specific location is located. For example, an antigen-binding molecule or its domain (e.g., a VH domain) containing an N-glycosylation site "within the C-terminal 10 amino acids" describes an antigen-binding molecule or its domain having the amino acid sequence NX[S / T] such that the Asn residue is within the C-terminal 10 amino acids of the antigen-binding molecule or its domain (e.g., a VH domain).

[0044] Operable connectionAs used herein, the term "operably linked" refers 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, or two or more nucleic acid sequences, for example, to produce an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence. In the context of fusion proteins or other polypeptides, the term "operably linked" refers to two or more amino acid segments being linked to produce a functional polypeptide. In the context of nucleic acids encoding fusion proteins (such as the antigen-binding molecules of this disclosure), "operably linked" means that two nucleic acids are linked such that the amino acid sequences encoded by the two nucleic acids remain within a reading frame. In the context of transcriptional regulation, the term refers to a functional relationship between a transcriptional regulatory sequence and a transcriptional sequence. For example, if a promoter or enhancer sequence stimulates or regulates transcription of a coding sequence in a suitable host cell or other expression system, then the promoter or enhancer sequence is operably linked to the coding sequence.

[0045] polypeptides, peptides and proteins The terms “polypeptide,” “peptide,” and “protein” are used interchangeably in this document and refer to polymers of amino acid residues.

[0046] Single-chain Fab or scFab As used herein, the term "single-chain Fab" or "scFab" refers to an ABD comprising a VH domain, a CH1 domain, a VL domain, a CL domain, and a linker. In some embodiments, the aforementioned domains and linker are arranged in one of the following orders in N-terminal to C-terminal orientation: (a) VH-CH1-linker-VL-CL, (b) VL-CL-linker-VH-CH1, (c) VH-CL-linker-VL-CH1, or (d) VL-CH1-linker-VH-CL. The linker is suitably an uncleavable linker of at least 30 amino acids, preferably between 32 and 50 amino acids. Single-chain Fab fragments are generally stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab molecules can be further stabilized by forming interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., at position 44 in the VH domain and position 100 in the VL domain according to Kabat numbering).

[0047] Single-chain Fv or scFvAs used herein, the term "single-chain Fv" or "scFv" refers to a polypeptide chain containing the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore (1994), Springer-Verlag, New York, pp. 269–315.

[0048] Subjects The term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cattle, chickens, amphibians, and reptiles. In some embodiments, the subject is a human. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably herein.

[0049] Quadrivalent As used herein, the term "tetravalent" refers to an antigen-binding molecule having four antigen-binding sites. In some embodiments, all four antigen-binding sites bind to the same epitope. In some embodiments, two of the antigen-binding sites bind to the same epitope, while the other two antigen-binding sites bind to different epitopes, regardless of whether these epitopes belong to the same or different target molecules. In other embodiments, two of the antigen-binding sites bind to the same epitope, a third antigen-binding site binds to a different epitope, and a fourth antigen-binding site binds to yet another different epitope. In still other embodiments, all four epitopes bind to different epitopes, regardless of whether these epitopes belong to the same or different target molecules. Thus, a tetravalent antigen-binding molecule can be monospecific, bispecific, trispecific, or tetraspecific.

[0050] Trivalent As used herein, the term "trivalent" refers to an antigen-binding molecule having three antigen-binding sites. In some embodiments, all three antigen-binding sites bind to the same epitope. In some embodiments, two of the antigen-binding sites bind to the same epitope, and the third antigen-binding site binds to a different epitope, whether these epitopes belong to the same target molecule or different target molecules. In other embodiments, all three antigen-binding sites bind to different epitopes, whether these epitopes are on the same target molecule or on any combination of two or more different target molecules. Thus, an antigen-binding molecule can be single-specific, bispecific, or trispecific.

[0051] Tumor-associated antigen (TAA) The term "tumor-associated antigen" or "TAA" refers to molecules that can be used to treat tumors when targeted by pharmacological agents. TAAs include cancer antigens as well as molecules expressed by or present on non-tumor cells that can be used to treat tumors, including, for example, extracellular matrix ("ECM") proteins, cell surface molecules of tumor or viral lymphocytes, T-cell antigens ("TCAs"), and immune checkpoint molecules.

[0052] Universal Light Chain, ULC As used in this article in the context of antigen-binding domains, the term "universal light chain" or "ULC" refers to a light chain polypeptide that can pair with the heavy chain region of the antigen-binding domain and also with other heavy chain regions. Universal light chains are also referred to as "shared light chains".

[0053] VH The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody (including the heavy chains of Fv, scFv, dsFv, or Fab).

[0054] VL The term "VL" refers to the variable region of the immunoglobulin light chain (including the light chains of Fv, scFv, dsFv, or Fab).

[0055] 6.2. Engineered Heavy Chain Variable Structural Domain

[0056] This disclosure provides a heavy chain variable domain (VH) whose primary amino acid sequence is modified to include an N-glycosylation site near the C-terminus (e.g., within 20, 10, 5, or 3 amino acids at the C-terminus).

[0057] Engineered VH domains can be incorporated into the antigen-binding domain (ABD) of an antigen-binding molecule. The ABD can be of any form, including forms such as scFv (e.g., as described in Section 6.3.1 below) and Fab (e.g., as described in Section 6.3.2 below), and can have various specificities, including TAA ABD (e.g., as described in Section 6.4.2 below) and T-cell adaptor (TCE) ABD (e.g., as described in Section 6.4.3 below). An exemplary scFv containing the engineered VH of this disclosure is depicted in... Figure 1A In the middle. An exemplary bivalent antigen-binding molecule containing two scFvs is depicted in Figure 1B In the diagram, two scFvs contain engineered VHs of this disclosure. Exemplary multivalent antigen-binding molecules containing engineered VHs of this disclosure are depicted in... Figure 1C middle.

[0058] The engineered heavy chain variable domain (VH; also referred to as "VH region" or "VH domain") disclosed herein typically contains (1) an N-glycosylation site within 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids at its C-terminus, or (2) an amino acid sequence at the C-terminus X1X2X3X4X5NX6X7X8X9X 10 X 11 X 12 (SEQ ID NO:1), where (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, optionally wherein the amino acid is not proline; (c) X7 is S or T, and (d) X8, X9, X 10 X 11 and X 12 Each is independently selected from: any amino acid and none. For convenience, such VHs are generally referred to as "engineered VHs" or "engineered VH domains" in this paper.

[0059] As described in this article, it has been found that modifying the VH in the antigen-binding domain, for example via amino acid substitution, insertion, or deletion, to incorporate an N-glycosylation site near the C-terminus of the VH (e.g., modifying the VH to include the sequence X1X2X3X4X5NX6[S / T]X8X9X at its C-terminus). 10 X 11 X 12 This leads to a decrease in the binding of antigen-binding molecules to antidrug antibodies (see, for example, section

[0219] (Example 4)). Therefore, this disclosure provides antigen-binding molecules comprising such engineered VH. Any antigen-binding molecule comprising VH can be modified such that VH has an N-glycosylation site near its C-terminus (e.g., the sequence X1X2X3X4X5NX6[S / T]X8X9X at its C-terminus). 10 X 11 X 12 ).

[0060] VHs can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), within which more conserved regions, called framework regions (FRs), are interspersed. Each VH 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. In some embodiments, the engineered VH of this disclosure includes an N-glycosylation site within the FR4 region. In other examples, the engineered VH of this disclosure includes an N-glycosylation site within the CDR3 region.

[0061] In some embodiments, the VH of this disclosure comprises the amino acid sequence X1X2X3X4X5NX6X7X8X9X at its C-terminus. 10 X 11 X 12 (SEQ ID NO:1), where (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, optionally wherein the amino acid is not proline; (c) X7 is S or T, and (d) X8, X9, X 10 X 11 and X 12 Each amino acid is independently selected from: any amino acid and the absence of amino acids. Such VH sequences may contain the sequence X1X2X3X4X5NX6[S / T]X8X9X. 10 X 11 X 12 This is because such sequences are inserted into the C-terminus of VH, or because one or more amino acid modifications are made to the original VH sequence to form the sequence X1X2X3X4X5NX6[S / T]X8X9X. 10 X 11 X 12 .

[0062] In some embodiments, X1 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, X2 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, X3 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, X4 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, X5 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, X6 is (i) A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y, or (ii) A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In some embodiments, X7 is S or T. In some embodiments, X8 is A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, or is not present. In some embodiments, X9 is A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, or is not present. In some embodiments, X 10The values ​​are A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, or none. In some embodiments, X 11 The values ​​are A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, or none. In some embodiments, X 12 The value is A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, or it does not exist.

[0063] In some respects, X8, X9, X 10 X 11 and X 12 One of them does not exist. In other respects, X8, X9, X 10 X 11 and X 12 Neither of these two exists. Furthermore, X8, X9, X 10 X 11 and X 12 None of the three are present. Furthermore, X8, X9, X 10 X 11 and X 12 None of the four are present. Furthermore, X8, X9, X 10 X 11 and X 12 None of the five are present.

[0064] Table 1 provides a complete list of possible amino acids at the C-terminal region of the engineered VH of this disclosure, wherein the VH contains the amino acid sequence X1X2X3X4X5XN6X7X8X9X at its C-terminus. 10 X 11 X 12 (SEQ ID NO:1). The amino acid sequence at the C-terminus of the engineered VH disclosed herein is X1X2X3X4X5NX6X7X8X9X. 10 X 11 X 12 It may contain any combination of amino acids at each position as outlined in Table 1.

[0065]

[0066] In some embodiments, X6 is selected from any amino acid other than proline.

[0067] In a particular embodiment, VH comprises at its C-terminus one of the following sequences (where X1, X2, X3, X4, and X5 are each independently selected from any amino acid): X1X2X3X4X5NSS (SEQ ID NO:2), X1X2X3X4X5NST (SEQ ID NO:3), X1X2X3X4VNSS (SEQ ID NO:4), X1X2X3X4VNST (SEQ ID NO:5), X1X2X3TVNSS (SEQ ID NO:6), X1X2X3TVNST (SEQ ID NO:7), X1X2VTVNSS (SEQ ID NO:8), X1X2VTVNST (SEQ ID NO:9), X1TVTVNSS (SEQ ID NO:10), X1TVTVNST (SEQ ID NO:11), TTVTVNSS (SEQ ID NO:12), TTVTVNST (SEQ ID NO:13), X1X2X3X4X5NSSGGGG (SEQ ID NO:14). ID NO:14), X1X2X3X4X5NSTGGGG (SEQ ID NO:15), X1X2X3X4VNSSGGGG (SEQ ID NO:16), NO:19), X1X2VTVNSSGGGG (SEQ ID NO:20), X1X2VTVNSTGGGG (SEQ ID NO:21), IDNO:25), X1X2X3X4X5NSSKPGG (SEQ ID NO:26), X1X2X3X4X5NSTKPGG (SEQ ID NO:27),X1X2X3VTNSSKPGG (SEQ ID NO:30), X1X2X3VTNSSKPGG (SEQ ID NO:31), X1X2VTVNSSKPGG (SEQ ID NO:32), NO:35), TTVTVNSSKPGG (SEQ ID NO:36), TTVTVNSTKPGG (SEQ ID NO:37), X1X2X3X4X5NSSPP (SEQ ID NO:38), X1X2X3X4X5NSTPP (SEQ ID NO:39), NO:40), X1X2X3X4VNSTPP (SEQ ID NO:41), X1X2X3TVNSSPP (SEQ ID NO:42), X1X2X3TVNSTPP (SEQ ID NO:43), X1X2VTVNSSPP (SEQ ID NO:44), NO:48) or TTVTVNSTPP (SEQ ID NO:49). ,

[0068] In some embodiments, VH contains the sequence VTVNSS (SEQ ID NO: 50) at its C-terminus. In some embodiments, VH contains the sequence VTVNST (SEQ ID NO: 51) at its C-terminus. In some embodiments, VH contains the sequence VTVNSSGGGG (SEQ ID NO: 52) at its C-terminus. In some embodiments, VH contains the sequence VTVNSTGGGG (SEQ ID NO: 53) at its C-terminus. In some embodiments, VH contains the sequence VTVNSSKPGG (SEQ ID NO: 54) at its C-terminus. In some embodiments, VH contains the sequence VTVNSTKPGG (SEQ ID NO: 55) at its C-terminus. In some embodiments, VH contains the sequence VTVNSSPP (SEQ ID NO: 56) at its C-terminus. In some embodiments, VH contains the sequence VTVNSTPP (SEQ ID NO: 57) at its C-terminus.

[0069] Without being bound by theory, human VHs are understood to typically contain the sequence VTVSS (SEQ ID NO:62) at their C-terminus. Therefore, aspects of this disclosure include modifying VHs containing the sequence VTVSS (SEQ ID NO:62) to include an N-glycosylation site at the C-terminus by inserting and / or substituting one or two amino acids within that sequence. For example, in one embodiment, the engineered VH of this disclosure is generated by inserting an N residue between the third and fourth positions of that sequence, thereby generating the sequence VTVNSS (SEQ ID NO:50) at the C-terminus. In another embodiment, the engineered VH of this disclosure is generated by inserting an N residue between the third and fourth positions of that sequence and also substituting a T residue at the fifth position, thereby generating the sequence VTVNST (SEQ ID NO:51).

[0070] In the context of an intact antigen-binding molecule, the sequence at the C-terminus of VH may be followed by any number of additional sequences, for example, in cases where VH is not at the C-terminus of the antigen-binding molecule. Alternatively, in some embodiments, such a sequence at the C-terminus of VH is also at the C-terminus of the antigen-binding molecule.

[0071] In addition to the N-glycosylation site, the VH of this disclosure may optionally further comprise one or more additional amino acid modifications or sequences designed to reduce binding to antidrug antibodies. Such additional modifications include, for example, the C-terminal sequence PP, the C-terminal sequence KPGG (SEQ ID NO: 66), V11K substitution (EU number), and the C-terminal polyglycine sequence (e.g., G4S (SEQ ID NO: 67)). The VH of this disclosure may comprise any one or more of these additional modifications, including any combination thereof.

[0072] The engineered VH domain of this disclosure can be incorporated into the antigen-binding domain of an antigen-binding molecule. Antigen-binding molecules containing the engineered VH of this disclosure are particularly described in section 6.4 and include, for example, various forms of antibodies, such as multivalent and / or multispecific antigen-binding molecules (e.g., as described in section 6.4.1.1 and section 6.4.1.2 below), antibody fragments, scFvs, and chimeric antigen receptors.

[0073] 6.3. Antigen-binding domain containing engineered VH domain

[0074] In some respects, engineered VH domains can be incorporated into ABDs, for example, into ABDs in antigen-binding molecules as described herein. In one embodiment, the ABD is an immunoglobulin molecule or a fragment thereof, particularly an IgG immunoglobulin molecule, and more particularly an IgG1 or IgG4 immunoglobulin molecule. Antibody fragments include, but are not limited to, VH fragments, VL fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, microantibodies, biantibodies, triantibodies, and tetraantibodies.

[0075] 6.3.1.scFv

[0076] In some embodiments, the engineered VH of this disclosure is incorporated into the scFv. In some embodiments, the VH of the scFv described herein may be an engineered VH. Exemplary scFvs incorporating engineered VHs are depicted in Figure 1A middle.

[0077] Single-chain Fv or “scFv” antibody fragments contain the VH and VL domains of the antibody within a single polypeptide chain, enabling expression as a single polypeptide chain while retaining the specificity of the complete antibody from which they originate. Generally, 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 those identified in Section 6.4.4.

[0078] Unless otherwise stated, the scFv used herein may have VL and VH variable regions in any order. For example, the scFv may contain VL-linker-VH or VH-linker-VL relative to the N-terminus and C-terminus of the polypeptide. For the avoidance of doubt, when the scFv of this disclosure contains an engineered VH, the engineered VH contains an N-glycosylation site (e.g., as a component of the amino acid sequence of SEQ ID NO:1) within three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20) amino acids at the C-terminus of the VH. Such N-glycosylation sites may be within three or more amino acids at the C-terminus of the scFv itself (e.g., where the scFv contains VL-linker-VH) or simply within three or more amino acids at the C-terminus of the VH, but further away from the C-terminus of the scFv (e.g., where the scFv contains VH-linker-VL).

[0079] scFv can contain VH and VL sequences from any suitable species, such as mice, humans, or humanized VH and VL sequences.

[0080] To generate scFv-encoded nucleic acids, DNA fragments encoding VH and VL are operatively ligated to another fragment encoding a linker, for example, any of the linkers described in Section 6.4.4, such as a repeat of a sequence containing the amino acids glycine and serine, so 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, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0081] 6.3.2.Fab

[0082] In some embodiments, the engineered VH of this disclosure is incorporated into the Fab.

[0083] Fab domains are traditionally generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. Fab domains can contain constant domains and variable region sequences from any suitable species, and therefore can be mouse, chimeric, human, or humanized.

[0084] The Fab domain typically contains 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 site. Disulfide bonds between the two constant domains further stabilize the Fab domain.

[0085] For the antigen-binding molecules of this disclosure, particularly when the light chains of ABDs are not common or universal light chains, it is advantageous to use a Fab heterodimerization strategy to allow proper association of Fab domains belonging to the same ABD and minimize aberrant pairing of Fab domains belonging to different ABDs. For example, the Fab heterodimerization strategies shown in Table H below can be used:

[0086]

[0087] 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.

[0088] Proper Fab pairing can also be facilitated by introducing one or more amino acid modifications into the CH1 domain of the Fab, one or more amino acid modifications into the CL domain of the Fab, and / or one or more amino acid modifications into the VH domain, and one or more amino acid modifications into 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 of other Fabs.

[0089] In one embodiment, one or more amino acid modifications are limited to conserved framework residues of variable domains (VH, VL) and constant domains (CH1, CL), as indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0090] In one embodiment, the modifications introduced into the VH and CH1 and / or VL and CL domains are complementary to each other. Complementarity at the heavy and light chain interfaces can be based on spatial and hydrophobic contacts, electrostatic / charge interactions, or combinations of various interactions. Complementarity between protein surfaces is widely described in the literature as lock-and-key mating, pestle-and-mortise, protrusions and cavities, donors and acceptors, etc., all of which suggest a structural and chemical matching nature between two interacting surfaces.

[0091] In one embodiment, one or more of the introduced modifications introduce new hydrogen bonds at the interface of the Fab component. In one embodiment, one or more of the introduced modifications introduce new salt bridges at the interface of the Fab component. Exemplary alternatives are described in WO 2014 / 150973 and WO 2014 / 082179, the contents of which are incorporated herein by reference.

[0092] In some embodiments, the Fab domain includes 192E substitution in the CH1 domain and 114A and 137K substitution in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, for example, Golay et al., 2016, JImmunol 196:3199-211).

[0093] In some embodiments, the Fab domain comprises 143Q and 188V substitutions in the CH1 domain and 113T and 176V substitutions in the CL domain, which are used to exchange the hydrophobic and polar regions of the contact between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0094] 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.

[0095] The Fab domains 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, MAbs 7:377-89).

[0096] The Fab domain can also be modified by replacing the CH1 and CL domains with alternative domains that promote proper assembly. For example, Wu et al., 2015, MAbs 7:364-76, described replacing the CH1 domain with the constant domain of the 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.

[0097] Instead of using a Fab heterodimerization strategy to facilitate proper VH-VL pairing, a shared light chain (also referred to as a universal light chain) VL can be used for each unique ABD in the antigen-binding molecules of this disclosure. In various embodiments, employing a shared light chain as described herein reduces the number of inappropriate species in the antigen-binding molecule compared to employing a pristine homologous VL. In various embodiments, the VL domain of the ABD is identified from monospecific antibodies containing the shared light chain. In various embodiments, the VH region of the ABD in the antigen-binding molecule contains 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 homologous to the human heavy chain or a single human light chain, and in response to exposure to the antigen of interest, generates an antibody library containing multiple human VHs homologous to one or two possible human VLs, wherein the antibody library is specific to the antigen of interest. The shared light chain is derived from rearranged human Vκ1-39Jκ5 sequences or rearranged human Vκ3-20Jκ1 sequences, and includes somatic mutant (e.g., affinity maturation) versions. See, for example, U.S. Patent No. 10,412,940.

[0098] 6.4. Antigen-binding molecules containing engineered VH domains

[0099] This disclosure provides an antigen-binding molecule comprising an engineered VH of this disclosure.

[0100] In some embodiments, the antigen-binding molecule is an antibody. The antibody can be any type of engineered antibody, including chimeric antibodies, humanized antibodies, veneered antibodies, or human antibodies. The antibody can be a monoclonal antibody or a genetically engineered polyclonal antibody. Antibodies considered herein include conventional antibodies as well as antibody-like molecules known in the art, including but not limited to nanobodies, biantibodies, microantibodies, antibody fragments, and other “alternative forms” of antibodies (e.g., as described in Spiess et al., 2015, MolImmunol, 67(2 Pt A):95-106, which is incorporated herein by reference). Antibodies can include engineered VHs of this disclosure as components of one or more ABDs, including, for example, as components of Fab or as components of scFv.

[0101] Therefore, in some embodiments, this document discloses an antibody comprising an engineered VH, the engineered VH comprising (1) an N-glycosylation site within 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids at its C-terminus, or (2) an amino acid sequence X1X2X3X4X5NX6X7X8X9X at its C-terminus. 10 X 11 X 12(SEQ ID NO:1), where (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, optionally wherein the amino acid is not proline; (c) X7 is S or T, and (d) X8, X9, X 10 X 11 and X 12 Each is independently selected from: any amino acid and none.

[0102] In some embodiments, the antibody of this disclosure comprises a polypeptide comprising: (a) VH, which includes (1) an N-glycosylation site within 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids at its C-terminus, or (2) an amino acid sequence X1X2X3X4X5NX6X7X8X9X at its C-terminus. 10 X 11 X 12 (SEQ ID NO:1); and (b) the Fc domain. The antibody may further comprise an additional polypeptide comprising an additional VH and an additional Fc domain. In some embodiments, the additional VH comprises (1) an N-glycosylation site within 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 amino acids at its C-terminus, or (2) an amino acid sequence X1X2X3X4X5NX6X7X8X9X at its C-terminus. 10 X 11 X 12 (SEQ ID NO:1). The antibody disclosed herein may contain any number of VHs (e.g., 1, 2, 3, 4 or more VHs), wherein at least one VH is an engineered VH as described herein, and wherein any number of additional VHs may or may not be engineered VHs as described herein.

[0103] 6.4.1.1. Multivalent antigen-binding molecules

[0104] The antigen-binding molecules disclosed herein include multivalent molecules having one or more engineered VH domains. Various forms of multivalent antigen-binding molecules are well-known in the art and are considered herein, including bivalent, trivalent, and tetravalent forms. Certain exemplary multivalent antigen-binding molecules are described in more detail below.

[0105] In some embodiments, an antigen-binding molecule comprising an engineered VH is a multivalent antigen-binding molecule comprising two or more scFvs linked via a linker, wherein at least one scFv comprises an engineered VH (as described, for example, in section 6.2 or as defined in numbered Examples 1 to 34). Thus, in some embodiments, antigen-binding molecules are disclosed herein comprising: (a) a first scFv, (b) a linker, and (c) a second scFv, wherein the first scFv and / or the second scFv comprises an engineered VH. In some embodiments, the first scFv comprises an engineered VH. In some embodiments, the second scFv comprises an engineered VH. In some embodiments, both the first scFv and the second scFv comprise an engineered VH. In some embodiments, the antigen-binding molecule further comprises a third scFv, which optionally comprises an engineered VH. Examples of such multivalent antigen-binding molecules comprising two scFvs include... Figure 1B The description.

[0106] In some embodiments, the antigen-binding molecule comprising an engineered VH is a multivalent antigen-binding molecule comprising three or more antigen-binding domains, wherein at least one of the antigen-binding domains comprises an engineered VH. Therefore, in some embodiments, an antigen-binding molecule is disclosed herein comprising: (a) a first polypeptide chain comprising, from its N-terminus to its C-terminus, (i) a first antigen-binding domain (e.g., Fab), (ii) a first dimerization moiety, and (iii) a second antigen-binding domain (e.g., scFv), wherein the second antigen-binding domain comprises an engineered VH; and (b) a second polypeptide chain comprising, from its N-terminus to its C-terminus, (i) a third antigen-binding domain (e.g., Fab), and (ii) a second dimerization moiety. In some embodiments, the second polypeptide further comprises a fourth antigen-binding domain (e.g., scFv) located at the C-terminus of the second dimerization moiety, wherein the fourth antigen-binding domain comprises an engineered VH. Examples of such multivalent antigen-binding molecules include... Figure 1C and Figures 2A to 2G As depicted in the text.

[0107] 6.4.1.2. Multispecific antigen-binding molecules

[0108] The antigen-binding molecules disclosed herein include multispecific molecules having one or more engineered VH domains. Various multispecific antigen-binding molecules are well-known in the art and are considered herein, including bispecific, trispecific, and tetraspecific antigen-binding molecules. Certain exemplary multispecific antigen-binding molecules are described in more detail below.

[0109] In some embodiments, the antigen-binding molecule comprising the engineered VH of this disclosure is a bispecific T-cell adaptor. A “bispecific T-cell adaptor” describes a molecule comprising two scFvs linked via a adapter, wherein the first scFv comprises a first ABD that binds to a T-cell antigen (e.g., the TCE ABD as described in section 6.4.3), and the second scFv comprises a second ABD that binds to a tumor-associated antigen (e.g., the TAA ABD as described in section 6.4.2). In some embodiments, the TCE ABD comprises an engineered VH. In some embodiments, the TAA ABD comprises an engineered VH. In some embodiments, both the TCE ABD and the TAA ABD comprise an engineered VH.

[0110] In some embodiments, the antigen-binding molecule comprising the engineered VH of this disclosure is a multivalent antigen-binding molecule comprising: (a) a first polypeptide chain comprising (i) a first TCE ABD or TAA ABD from its N-terminus to its C-terminus, (ii) a first dimerization moiety, and (iii) a second TCE ABD or TAA ABD; and (b) a second polypeptide chain comprising (i) a third TCE ABD or TAA ABD from its N-terminus to its C-terminus, and (ii) a second dimerization domain, wherein the second TCE ABD or TAA ABD comprises the engineered VH. In some embodiments, the second TCE ABD or TAA ABD is an scFv. In some embodiments, the second polypeptide further comprises a TCE ABD or TAA ABD located at the C-terminus of the second dimerization moiety, wherein a fourth TCE ABD or TAA ABD comprises the engineered VH. In some embodiments, the fourth TCE ABD or TAA ABD is an scFv.

[0111] In some embodiments, the first ABD is a TCE ABD (e.g., as described in section 6.4.3). In some embodiments, the first ABD is a TAA ABD (e.g., as described in section 6.4.2). In some embodiments, the second ABD is a TCE ABD (e.g., as described in section 6.4.3). In some embodiments, the second ABD is a TAA ABD (e.g., as described in section 6.4.2). In some embodiments, the third ABD is a TCE ABD (e.g., as described in section 6.4.3). In some embodiments, the third ABD is a TAA ABD (e.g., as described in section 6.4.2). In some embodiments, the fourth ABD is a TCE ABD (e.g., as described in section 6.4.3). In some embodiments, the fourth ABD is a TAA ABD (e.g., as described in section 6.4.2).

[0112] 6.4.2. Tumor-associated antigen ABD

[0113] In some embodiments, the antigen-binding molecule of this disclosure comprises at least one ABD that specifically binds to a tumor-associated antigen (TAA), referred to herein as “TAA ABD”. Examples of TAAs include cancer antigens, extracellular matrix (“ECM”) proteins, tumor-reactive lymphocyte antigens, cell surface molecules of tumor or viral lymphocytes, T-cell antigens (“TCAs”), and immune checkpoint molecules. Those skilled in the art will recognize that the foregoing categories of target molecules are not mutually exclusive, and thus a given target molecule may fall into more than one of the foregoing categories. For example, some molecules may be considered both TCAs and immune checkpoint molecules. Preferably, the TAA is a human antigen. The antigen may or may not be present on normal cells. Certain aspects involve antigen-binding molecules comprising at least one ABD that specifically binds to a TAA. In some embodiments, the TAA is preferentially expressed or upregulated on tumor cells compared to normal cells. In other embodiments, the TAA is a lineage marker.

[0114] It is anticipated that any type of tumor and any type of TAA can be targeted by the antigen-binding molecules disclosed herein. Exemplary cancer types that can be targeted include acute lymphoblastic leukemia, acute myeloid leukemia, cholangiocarcinoma, B-cell leukemia, B-cell lymphoma, cholangiocarcinoma, bone cancer, brain cancer, breast cancer, triple-negative breast cancer, cervical cancer, Burkitt lymphoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, glioma, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, medullary thyroid carcinoma, melanoma, multiple myeloma, ovarian cancer, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, lung cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, urothelial carcinoma, and other bladder cancers. However, those skilled in the art will recognize that TAAs are known for virtually any type of cancer.

[0115] Non-limiting examples of ECM antigens include multiligand proteoglycans, 27enzalkoni, integrins, osteopontin, connexins, cadherins, laminin, laminin-type EGF, lectins, fibronectin, notch, adhesion proteins (e.g., adhesion protein-4), tendinogen, collagen (e.g., type X collagen), and matrix metalloproteinases.

[0116] Other target molecules are cell surface molecules of tumor or viral lymphocytes, such as T cell costimulatory proteins like CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0117] In certain embodiments, the target molecule is an immune checkpoint molecule, such as CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, and CHK2. In a specific embodiment, the target molecule is PD1. In other embodiments, the target molecule is LAG3. In some embodiments, when the target molecule is an immune checkpoint molecule, TAA ABD is non-blocking or weakly blocking ligand-receptor binding. Examples of non-blocking or weakly blocking anti-PD1 antibodies include antibodies with the VH / VL amino acid sequence of SEQ ID No: 2 / 10 of PCT Publication No. WO2015 / 112800A1; SEQ ID No: 16 / 17 of U.S. Patent No. 11,034,765 B2; and antibodies with the VH / VL amino acid sequences of SEQ ID Nos. 164 / 178, 165 / 179, 166 / 180, 167 / 181, 168 / 182, 169 / 183, 170 / 184, 171 / 185, 172 / 186, 173 / 187, 174 / 188, 175 / 189, 176 / 190, and 177 / 190 of U.S. Patent No. 10,294,299 B2. Examples of non-blocking or weakly blocking anti-LAG3 antibodies include antibodies having the VH / VL amino acid sequences of SEQ ID NO 23 / 24, ¾, and 11 / 12 as disclosed in US2022 / 0056126A1.

[0118] In some embodiments, the target molecule is a TAA. Exemplary TAAs are listed in Table A below, along with references to exemplary antibodies or antibody sequences on which TAA ABDs may be based.

[0119]

[0120] In some aspects, the TAA ABD competes with the antibodies listed in Table A for binding to TAA. In a further aspect, the TAA ABD comprises a CDR having the CDR sequence of the anti-TAA antibody listed in Table A. In some embodiments, the TAA ABD comprises all six CDR sequences of the anti-TAA antibody listed in Table A. In other embodiments, the TAA ABD comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of the anti-TAA antibody listed in Table A and the light chain CDR sequence of the universal light chain. In a further aspect, the TAA ABD comprises a VH containing the amino acid sequence of the VH of the anti-TAA antibody listed in Table A. In some embodiments, the TAA ABD further comprises a VL containing the amino acid sequence of the VL of the anti-TAA antibody listed in Table A. In other embodiments, the TAA ABD further comprises a universal light chain VL sequence.

[0121] Other TAAs that can be targeted by antigen-binding molecules are disclosed, for example, in Hafeez et al., 2020, Molecules 25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. The entire contents of Table 1 by Hafeez et al. are incorporated herein by reference.

[0122] Other exemplary TAAs include fibroblast activator protein (FAP), the A1 domain of tendinin-C (TNCA1), the A2 domain of tendinin-C (TNC A2), the extra domain B of fibronectin (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, and dipeptidyl peptidase IV. (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-ζ chain, and the MAGE family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4). (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), the GAGE ​​family of tumor antigens (e.g.,GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100, Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis protein (APC), fodrin, connexin. 37. Ig idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, Smad tumor antigen family, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (heteromeric multimer associated with TCR), CD22 (B cell receptor), CD23 (low-affinity IgE receptor), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R- (IL6 receptor), CD20, MCSP, PDGFβR (β-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disialiacoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muco-hsp70-2, M-CSF, prostaglandins, prostaglandin-specific antigen (PSA), PAP, LAGA-1a, p53, prostaglandin, PSMA, susceptin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, hepatin B2, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor matrix antigen, extra domain A (EDA) and extra domain B of fibronectin. (EDB) and the A1 domain of tendin-C (TnC A1).

[0123] 6.4.3.TCE ABD

[0124] In some embodiments, the antigen-binding molecule of this disclosure comprises at least a T-cell conjugating ABD that specifically binds to a component of the T-cell receptor complex, referred to herein as "TCE ABD". In other embodiments, the antigen-binding molecule of this disclosure does not comprise a TCE ABD. Exemplary targets of the TCE ABD are CD3 and T-cell receptors (e.g., TCRαβ or TCRγδ). Preferably, the TCE ABD targets a component of the human T-cell receptor complex. The epitope of the TCE ABD may be a single polypeptide (e.g., CD3ε) or a multimeric component of the T-cell receptor complex (e.g., a TCRαβ dimer or a TCRγδ dimer).

[0125] Exemplary CD3 and TCR antibodies or antibody sequences are listed in Table T below, and TCE ABD can be based on these antibodies or antibody sequences.

[0126]

[0127] In some aspects, the TCE ABD competes with the T cell conjugating (TCE) antibodies listed in Table T for binding to a target of the TCE antibody (e.g., CD3 or T cell receptor). In a further aspect, the TCE ABD comprises a CDR having the CDR sequence of the TCE antibody listed in Table T. In some embodiments, the TCE ABD comprises all six CDR sequences of the TCE antibody listed in Table T. In other embodiments, the TCE ABD comprises at least the heavy chain CDR sequences (CDR-H1, CDR-H2, CDR-H3) of the TCE antibody listed in Table T and the light chain CDR sequence of the universal light chain. In a further aspect, the TCE ABD comprises a VH containing the amino acid sequence of the VH of the TCE antibody listed in Table T. In some embodiments, the TCE ABD further comprises a VL containing the amino acid sequence of the VL of the TCE antibody listed in Table T. In other embodiments, the TCE ABD further comprises a universal light chain VL sequence.

[0128] 6.4.4. Connector

[0129] In some aspects, this disclosure provides recombinant polypeptides wherein two or more components of the recombinant polypeptide are linked to each other by a linker (also referred to as a “peptide linker”). By way of example and not limitation, the linker may be used to link (a) a target-binding domain and a constant domain; (b) a first target-binding domain and a second target-binding domain (e.g., a first scFv and a second scFv); or (c) different domains within the target-binding domain (e.g., VH and VL domains in the scFv).

[0130] The length of the peptide linker can range from 2 amino acids to 60 or more amino acids, and in some respects, the length of the peptide linker ranges from 3 amino acids to 50 amino acids, 4 amino acids to 30 amino acids, 5 amino acids to 25 amino acids, 10 amino acids to 25 amino acids, 10 amino acids to 60 amino acids, 12 amino acids to 20 amino acids, 20 amino acids to 50 amino acids, or 25 amino acids to 35 amino acids.

[0131] In certain respects, the length of the peptide linker is at least 5 amino acids, at least 6 amino acids, or at least 7 amino acids, and optionally, the length is at most 30 amino acids, at most 40 amino acids, at most 50 amino acids, or at most 60 amino acids.

[0132] In some of the foregoing embodiments, the length of the connector ranges from 5 to 50 amino acids, for example, from 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids. In other foregoing embodiments, the length of the connector ranges from 6 to 50 amino acids, for example, from 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids. In yet other foregoing embodiments, the length of the connector ranges from 7 to 50 amino acids, for example, from 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids.

[0133] Charged (e.g., charged hydrophilic connectors) and / or flexible connectors are particularly preferred.

[0134] Examples of flexible linkers that can be used for the recombinant peptides of this disclosure include those disclosed by Chen et al., 2013, Adv DrugDeliv Rev. 65(10): 1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are monomers or polymers comprising repeating sequences of glycine and serine, such as GnS (SEQ ID NO: 68) or SGn (SEQ ID NO: 69), where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is a monomer or polymer comprising a repeating sequence of G4S (SEQ ID NO: 67), such as (GGGGS). n , where n is an integer from 1 to 10 (SEQ ID NO: 70), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in some embodiments, the connector is GGGGS (SEQ ID NO: 67), GGGGSGGGGS (SEQ ID NO: 71), GGGGSGGGGSGGGGS (SEQ ID NO: 72), GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 73), or GGGGSGGGGSGGGGSGGGSGGGSGGGS (SEQ ID NO: 74).

[0135] Polyglycine linkers may be suitably used in the recombinant peptides of this disclosure. In some embodiments, the peptide linker comprises two consecutive glycines (2 Gly), three consecutive glycines (3 Gly), four consecutive glycines (4 Gly), five consecutive glycines (5 Gly), six consecutive glycines (6 Gly), seven consecutive glycines (7 Gly), eight consecutive glycines (8 Gly), or nine consecutive glycines (9 Gly).

[0136] 6.4.5. Fc area

[0137] In some respects, the antigen-binding molecule of this disclosure comprises a pair of Fc domains that associate to form an Fc region. In natural antibodies, the Fc region includes a hinge region at its N-terminus to form a constant domain. Throughout this disclosure, unless otherwise stated, references to Fc domains cover Fc domains having a hinge domain at their N-terminus.

[0138] The Fc domain can be derived from any suitable species that is operatively linked to an ABD or a component thereof. In one embodiment, the Fc domain is derived from a human Fc domain. In a preferred embodiment, the antigen-binding domain of the antigen-binding molecule of this disclosure is fused to an IgG Fc molecule. The antigen-binding domain may be fused to the N-terminus or C-terminus of the IgG Fc domain, or both.

[0139] The Fc domain can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0140] The two Fc domains within the Fc region may be identical or different from each other. In natural antibodies, the Fc domains are typically identical, but for the purpose of generating multispecific binding molecules, such as the antigen-binding molecules described herein, the Fc domains may advantageously be different to allow heterodimerization, as described in Section 6.4.5.2 below. In other embodiments, the two Fc domains of the antigen-binding molecules disclosed herein are identical.

[0141] 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 the Fc region.

[0142] In the antigen-binding molecules disclosed herein, the Fc region and / or the Fc domain within it may contain heavy chain constant domains from one or more different classes of antibodies, such as one, two or three different classes.

[0143] In one embodiment, the Fc region contains CH2 and CH3 domains derived from IgG1.

[0144] In one embodiment, the Fc region contains CH2 and CH3 domains derived from IgG2.

[0145] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG3.

[0146] In one embodiment, the Fc region contains CH2 and CH3 domains derived from IgG4.

[0147] In one embodiment, the Fc region contains a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain.

[0148] In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG and a CH4 domain derived from IgM.

[0149] It should be understood that the heavy chain constant domain of the Fc region used to generate the antigen-binding molecule of this disclosure may include variants of the aforementioned naturally occurring constant domains. Such variants may contain one or more amino acid variations compared to the wild-type constant domain. In one instance, the Fc region of this disclosure contains at least one constant domain whose sequence differs 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.

[0150] IgM and IgA are naturally occurring covalent polymers of common H2L2 antibody units in the human body. When IgM is incorporated into the J chain, it appears as a pentamer; or when the J chain is absent, it appears as a hexamer. IgA occurs in both monomeric and dimeric forms. The heavy chains of IgM and IgA have an 18-amino acid extension with a constant C-terminal domain, called a tail. The tail contains cysteine ​​residues, which form disulfide bonds between the heavy chains of the polymer and are considered to play an important role in polymerization. The tail also contains glycosylation sites. In some embodiments, the antigen-binding molecules of this disclosure do not contain a tail.

[0151] The Fc domain incorporated into the antigen-binding molecule of this disclosure may include one or more modifications that alter the functional properties of the protein, such as binding to Fc receptors such as 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 6.4.5.1.

[0152] The Fc domain can also be modified to include modifications that improve the manufacturability of asymmetric antigen-binding molecules, for example, by allowing heterodimerization, which is the pairing of different Fc domains over the same Fc domain. Heterodimerization allows the generation of antigen-binding molecules in which different polypeptide components are linked to each other through Fc regions containing Fc domains with different sequences. Examples of heterodimerization strategies are illustrated in Section 6.4.5.2.

[0153] 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 alter the properties of the antigen-binding molecule.

[0154]

[0155] In some respects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 58. Where the Fc domain comprises an amino acid sequence having at least 90% sequence identity and less than 100% sequence identity with SEQ ID NO: 58 (e.g., sequence identity between 90% and 99% with SEQ ID NO: 58), the Fc domain may also comprise one or more amino acid substitutions as described herein, such as one or more substitutions that reduce effector function (e.g., as described in section 6.4.5.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in section 6.4.5.2).

[0156] In some respects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 59. Where the Fc domain comprises an amino acid sequence having at least 90% sequence identity and less than 100% sequence identity with SEQ ID NO: 59 (e.g., sequence identity between 90% and 99% with SEQ ID NO: 59), the Fc domain may also comprise one or more amino acid substitutions as described herein, such as one or more substitutions that reduce effector function (e.g., as described in section 6.4.5.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in section 6.4.5.2).

[0157] In some respects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 60. Where the Fc domain comprises an amino acid sequence having at least 90% sequence identity and less than 100% sequence identity with SEQ ID NO: 60 (e.g., sequence identity between 90% and 99% with SEQ ID NO: 60), the Fc domain may also comprise one or more amino acid substitutions as described herein, such as one or more substitutions that reduce effector function (e.g., as described in section 6.4.5.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in section 6.4.5.2).

[0158] In some respects, the Fc domain comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 61. Where the Fc domain comprises an amino acid sequence having at least 90% sequence identity and less than 100% sequence identity with SEQ ID NO: 61 (e.g., sequence identity between 90% and 99% with SEQ ID NO: 61), the Fc domain may also comprise one or more amino acid substitutions as described herein, such as one or more substitutions that reduce effector function (e.g., as described in section 6.4.5.1) and / or one or more substitutions that promote Fc heterodimerization (e.g., as described in section 6.4.5.2).

[0159] 6.4.5.1. Fc domain with altered effector function

[0160] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.

[0161] 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 activating Fc receptor. In a particular embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is selected from one or more of the group consisting 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.

[0162] In one embodiment, the Fc domain (e.g., the Fc domain of an antigen-binding molecule) or the Fc region (e.g., one or both Fc domains of an antigen-binding molecule that can associate to form an Fc region) contains an amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297, P331, and P329 (according to Kabat EU index numbers). In a more specific embodiment, the Fc domain or Fc region contains an amino acid substitution at a position selected from L234, L235, and P329 (according to Kabat EU index numbers). In some embodiments, the Fc domain or Fc region contains amino acid substitutions L234A and L235A (according to Kabat EU index numbers). In one such embodiment, the Fc domain or region is an Igd Fc domain or region, particularly a human Igd Fc domain or region. In one embodiment, the Fc domain or Fc region contains an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (according to the Kabat EU index number). In one embodiment, the Fc domain or Fc region comprises an amino acid substitution at position P329 and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (according to the Kabat EU index number). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain or Fc region comprises amino acid substitutions at positions P329, L234, and L235 (according to the Kabat EU index number). In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A, and P329G (“P329G LALA”, “PGLALA”, or “LALAPG”).

[0163] Typically, each of the two Fc domains in the Fc region contains the same one or more amino acid substitutions. Therefore, in a specific embodiment, each Fc domain of the Fc region contains 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 substituted with an alanine residue (L234A), the leucine residue at position 235 is substituted with an alanine residue (L235A), and the proline residue at position 329 is substituted with a glycine residue (P329G) (according to the Kabat EU index number).

[0164] In one embodiment, the Fc domain is the IgG1 Fc domain, particularly the human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing D265A, N297A mutations (EU number) to reduce effector function.

[0165] 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 comprise an amino acid sequence selected from Table F-2 below. In some embodiments, the Fc domain includes only the bolded portion of the sequence shown below:

[0166]

[0167] In certain embodiments, the effector-reduced IgG4 comprises the bold portion of the amino acid sequence of SEQ ID NO:31 of WO2014 / 121087, sometimes referred to herein as IgG4 or hIgG4.

[0168] For the heterodimer Fc region, combinations of the above-mentioned variant IgG4 Fc sequences can be incorporated, such as an Fc region containing an Fc domain of the amino acid sequence (or its bolded portion) of SEQ ID NO:30 of WO2014 / 121087 and an Fc domain containing an amino acid sequence (or its bolded portion) of SEQ ID NO:37 of WO2014 / 121087, or an Fc region containing an Fc domain of the amino acid sequence (or its bolded portion) of SEQ ID NO:31 of WO2014 / 121087 and an Fc domain containing an amino acid sequence (or its bolded portion) of SEQ ID NO:38 of WO2014 / 121087.

[0169] 6.4.5.2. Fc heterodimer variant

[0170] Some antigen-binding molecules require dimerization between two Fc domains. Unlike natural immunoglobulins, the two Fc domains are operatively linked to different N-terminal or C-terminal regions. Insufficient heterodimerization of the two Fc domains to form the Fc region can be a barrier to increasing the yield of the desired heterodimer molecule and poses a challenge to purification. Various methods available in the art can be used to enhance the dimerization of the Fc domains that may be present in the antigen-binding molecules disclosed herein, such as those disclosed in: EP 1870459A1; U.S. Patent No. 5,582,996; U.S. Patent No. 5,731,168; U.S. Patent No. 5,910,573; U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441; U.S. Patent No. 7,183,076; U.S. Patent Application Publication No. 2006204493A1; and PCT Publication No. WO 2009 / 089004A1.

[0171] In some embodiments, this disclosure provides an antigen-binding molecule comprising an Fc heterodimer, i.e., an Fc region containing heterologous, distinct Fc domains. Typically, each Fc domain in the Fc heterodimer contains a CH3 domain of an antibody. The CH3 domain is derived from a constant region of any isotype, class, or subclass of antibody, and preferably from a constant region of antibodies of the IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the previous section.

[0172] In specific embodiments, the modification that promotes Fc heterodimer formation is a so-called "mortar and pestle" modification, comprising a "mortar" modification in one Fc domain and a "pestle" modification in the other Fc domain. Mortar and pestle techniques are described, for example, in U.S. Patent Nos. 5,731,168; US 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621; and Carter, 2001, Immunol Meth 248:7-15. Generally, the method involves introducing a protrusion ("pestle") at the interface of a first polypeptide and a corresponding cavity ("mortar") at the interface of a second polypeptide, such that the protrusion can be positioned within the cavity, thereby promoting heterodimer formation and inhibiting homodimer formation. The protrusion is constructed by replacing the small amino acid side chains from the first polypeptide interface with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensating cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide.

[0173] Therefore, in some embodiments, amino acid residues in the CH3 domain of the first subunit of the Fc domain are substituted with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain of the first subunit. This protrusion can be positioned in a cavity within the CH3 domain of the second subunit. Conversely, amino acid residues in the CH3 domain of the second subunit of the Fc domain are substituted with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit. The protrusion within the CH3 domain of the first subunit can be positioned within this cavity. Preferably, the amino acid residues with a larger side chain volume are selected from the group consisting of arginine (I), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residues with a smaller side chain volume are selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, through site-specific mutagenesis or peptide synthesis. An exemplary substitution is Y470T.

[0174] In a specific embodiment of this kind, in the first Fc domain, the threonine residue at position 366 is substituted with a tryptophan residue (T366W), and in the Fc domain, the tyrosine residue at position 407 is substituted with a valine residue (Y407V). Optionally, the threonine residue at position 366 is substituted with a serine residue (T366S), and the leucine residue at position 368 is substituted with an alanine residue (L368A) (according to the Kabat EU index number). In a further embodiment, in the first Fc domain, the serine residue at position 354 is additionally substituted with a cysteine ​​residue (S354C), or the glutamate residue at position 356 is substituted with a cysteine ​​residue (E356C) (particularly the serine residue at position 354 is substituted with a cysteine ​​residue), and in the second Fc domain, the tyrosine residue at position 349 is additionally substituted with a cysteine ​​residue (Y349C) (according to the Kabat EU index number). In a particular embodiment, the first Fc domain comprises amino acid substitutions S354C and T366W, and the second Fc domain comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (according to Kabat EU index numbers).

[0175] In some embodiments, electrostatic redirection (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25): 19637-46) can be used to facilitate the bonding of the first and second Fc domains in the Fc region.

[0176] As an alternative or 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 polypeptide comprises a modified Fc domain that eliminates its binding to protein A, thereby enabling purification methods that produce heterodimeric proteins. See, for example, U.S. Patent No. 8,586,713. Thus, an antigen-binding molecule comprises a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein at least one amino acid difference reduces the binding of the antigen-binding molecule to protein A compared to a corresponding antigen-binding molecule lacking an amino acid difference. In one embodiment, the first CH3 domain binds protein A and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as an H95R modification (via IMGT exon number; H435R via EU number). The second CH3 may further comprise a Y96F modification (via IMGT; Y436F via EU). Such modifications are referred to herein as “star” mutations.

[0177] In some embodiments, Fc may contain one or more mutations (e.g., pestle and mortar mutations) to promote heterodimerization and star mutations to promote purification.

[0178] 6.4.5.3. Hinge Structural Domain

[0179] The antigen-binding molecule of this disclosure may include an Fc domain containing a hinge domain at its N-terminus. The hinge region may be natural or modified. The hinge region is typically located at the N-terminus of the Fc region. Unless the context otherwise specifies, the term "hinge domain" refers to a naturally occurring or non-naturally occurring hinge sequence that is a monomeric hinge domain in the case of a single or monomeric polypeptide chain, and may be contained as two associated hinge sequences on a single polypeptide chain in the case of a dimer polypeptide (e.g., a homodimer or heterodimer antigen-binding molecule formed by the association of two Fc domains). Sometimes, the two associated hinge sequences are referred to as "hinge regions." In some embodiments of the antigen-binding molecule of this disclosure, further iterations of the hinge region may be incorporated into the polypeptide sequence.

[0180] A natural hinge region is a hinge region typically found between the Fab and Fc domains of a naturally occurring antibody. A modified hinge region is any hinge that differs from a natural hinge region in length and / or composition. Such hinges can include hinge regions derived from other species, such as those of humans, mice, rats, rabbits, sharks, pigs, hamsters, camels, llamas, or goats. Other modified hinge regions may comprise complete hinge regions derived from antibodies of a different class or subclass than the heavy chain Fc domain or Fc region. Alternatively, a modified hinge region may comprise a portion of a natural hinge or repeating unit, where each unit in the repeat is derived from a natural hinge region. In a further alternative, a natural hinge region can be altered by converting one or more cysteine ​​or other residues to neutral residues, such as serine or alanine, or by converting appropriately placed residues to cysteine ​​residues. In this 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 designed to have desired properties, such as length, cysteine ​​composition, and flexibility.

[0181] 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, and these contents are incorporated herein by reference.

[0182] In one embodiment, the antigen-binding molecule of this disclosure comprises an Fc region, wherein one or both Fc domains have a fully hinged domain at their N-terminus.

[0183] In various embodiments, positions 233-236 within the hinge area can be G, G, G and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, wherein the positions are numbered by EU numbering.

[0184] In some embodiments, the antigen-binding molecule of this disclosure includes a modified hinge region that has reduced binding affinity to the Fcγ receptor relative to the wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0185] In one embodiment, the antigen-binding molecule of this disclosure comprises an Fc region, wherein each Fc domain has a fully hinged domain at its N-terminus, wherein each Fc domain and hinged domain is derived from IgG4 and each hinged domain contains the modified sequence CPPC (SEQ ID NO: 87). Compared to IgG1 containing the sequence CPPC (SEQ ID NO: 87), the core hinge region of human IgG4 contains the sequence CPSC (SEQ ID NO: 88). The presence of serine residues in the IgG4 sequence increases the flexibility of this region, allowing a portion of the molecule to form disulfide bonds within the same protein chain (intra-chain disulfide bonds) rather than bridging to other heavy chains in the IgG molecule to form inter-chain disulfide bonds (Angel et al., 1993, Mol Immunol 30(1):105-108). Replacing serine residues with proline to obtain the same core sequence as IgG1 allows for complete inter-chain disulfide bond formation in the IgG4 hinge region, thereby reducing heterogeneity in the purified product. This modified isotype is referred to as IgG4P.

[0186] 6.4.5.3.1. Interlocking Hinge Sequence

[0187] The hinge structure domain can be a mating hinge structure domain (also known as a "matting hinge area").

[0188] For example, the chimeric hinge domain may contain 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.

[0189] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 89) (previously disclosed as SEQ ID NO: 8 of WO2014 / 121087, which is incorporated herein by reference in its entirety) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 90) (previously 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 incorporating an IgG4 Fc domain, such as a human or mouse Fc domain, which may be further modified in the CH2 and / or CH3 domains to reduce effector function, for example as described in section 6.4.5.1).

[0190] 6.4.5.3.2. Hinge sequences with reduced effector functionality

[0191] In a further embodiment, the hinge region may be modified to reduce the effector function, for example as described in WO2016161010A2, the entirety of which is incorporated herein by reference. In various embodiments, the modified hinge region positions 233-236 are G, G, G and unoccupied; G, G, unoccupied and unoccupied; G, unoccupied, unoccupied and unoccupied; or all unoccupied, wherein the positions are numbered by EU numbering (as shown in Figure 1 of WO2016161010A2). These segments may be represented as GGG-, GG--, G---, or ----, where "-" indicates an unoccupied position.

[0192] Position 236 is not occupied in canonical human IgG2, but is occupied in other classic human IgG isotypes. In all four human isotypes, positions 233-235 are occupied by residues other than G (as shown in Figure 1 of WO2016161010A2).

[0193] 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 antibodies is beneficial for stabilizing IgG4 antibodies 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.

[0194] 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 without G(233-236). Optionally, the hinge domain amino acid sequence includes CPCPAPGGG-GPSVF (SEQ ID NO: 91) (SEQ ID NO: 1 previously disclosed as WO2016161010A2), CPCPAPGG-GPSVF (SEQ ID NO: 92) (SEQ ID NO: 2 previously disclosed as WO2016161010A2), CPCPAPG---GPSVF (SEQ ID NO: 93) (SEQ ID NO: 3 previously disclosed as WO2016161010A2), or CPCPAPG----GPSVF (SEQ ID NO: 94) (SEQ ID NO: 4 previously disclosed as WO2016161010A2).

[0195] The modified hinge region described above can be incorporated into the heavy chain constant region, which typically includes CH2 and CH3 domains, and may have additional hinge segments (e.g., upper hinges) located on the flanks of the designated region. Such additional constant region segments generally have the same isotype, preferably a human isotype, although they may be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may also be human IgG1, IgG2, or IgG3, or hybrids of their domains with different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2 to 4 of WO2016161010A2.

[0196] In a specific embodiment, the modified hinge sequence may be linked to the IgG4 CH2 region (e.g., by incorporating 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, for example as described in section 6.4.5.1).

[0197] 6.5. Nucleic Acids and Host Cells

[0198] On the other hand, this disclosure provides nucleic acids that encode the antigen-binding molecules of this disclosure. In some embodiments, the antigen-binding molecule is encoded by a single nucleic acid. In other embodiments, such as in the case of heterodimeric molecules or molecules comprising components consisting of more than one polypeptide chain, the antigen-binding molecule may be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0199] A single nucleic acid can encode an antigen-binding molecule containing a single polypeptide chain, an antigen-binding molecule containing two or more polypeptide chains, or a portion of an antigen-binding molecule containing more than two polypeptide chains (e.g., a single nucleic acid can encode two polypeptide chains of an antigen-binding molecule containing three, four, or more polypeptide chains, or three polypeptide chains of an antigen-binding molecule containing 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 element and separated by an internal ribosome entry site (IRES) sequence, thereby allowing translation into individual polypeptides.

[0200] In some embodiments, an antigen-binding molecule comprising two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the antigen-binding molecule may be equal to or less than the number of polypeptide chains in the antigen-binding molecule (e.g., when more than one polypeptide chain is encoded by a single nucleic acid).

[0201] The nucleic acid disclosed herein can be DNA (e.g., plasmid) or RNA (e.g., mRNA).

[0202] In another aspect, this disclosure provides host cells and vectors containing the nucleic acids of this disclosure. The nucleic acids may be present in a single vector or in a single vector within the same host cell or a separate host cell, as described in more detail below.

[0203] 6.6. Pharmaceutical Compositions

[0204] The antigen-binding molecule disclosed herein may be in the form of a composition comprising an antigen-binding molecule and one or more carriers, excipients, and / or diluents. The composition may be formulated for a specific purpose, such as for veterinary or human pharmaceutical 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 antigen-binding molecule and the mode of administration for therapeutic purposes.

[0205] 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 method required for its 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, topically, or locally. The most appropriate route of administration in any given situation will depend on the specific antibody, 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.

[0206] Pharmaceutical compositions can be conveniently available in unit dosage forms containing a predetermined amount of the antigen-binding molecule of this disclosure per dose. The amount of antigen-binding molecule contained in a 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 antigen-binding molecule 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 amount of diluent, and / or other components for administration. The liquid unit dose can be conveniently supplied in the form of a syringe pre-filled with a predetermined amount of antigen-binding molecule suitable for a single administration.

[0207] The pharmaceutical composition can also be supplied in bulk from a certain amount of antigen-binding molecules suitable for multiple administrations.

[0208] Pharmaceutical compositions can be prepared by mixing an antigen-binding molecule 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 various other 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 used.

[0209] Buffers help maintain pH values ​​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 succinate and monosodium succinate, mixtures of succinate and sodium hydroxide, mixtures of succinate 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, mixtures of fumaric acid and sodium fumarate, etc.). Mixtures of fumarate-disodium fumarate, monosodium fumarate-disodium fumarate, etc., gluconate buffers (e.g., mixtures of gluconate-sodium gluconate, gluconate-sodium hydroxide, gluconate-potassium gluconate, etc.), oxalate buffers (e.g., mixtures of oxalate-sodium oxalate, oxalate-sodium hydroxide, oxalate-potassium oxalate, etc.), lactate buffers (e.g., mixtures of lactate-sodium lactate, lactate-sodium hydroxide, lactate-potassium lactate, etc.), and acetate buffers (e.g., mixtures of acetate-sodium acetate, acetate-sodium hydroxide, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts (such as Tris) can also be used.

[0210] Preservatives may be added to delay microbial growth, and may be added in amounts ranging from about 0.2% to 1% (w / v). Preservatives suitable for use in this disclosure include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, 59-benzalkonium halides (e.g., chlorides, bromides, and iodides), hexamethylammonium chloride, and alkylparaben esters (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 isotonic agents include polyols, such as ternary or higher sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a large class of excipients whose functional range includes fillers to additives, capable of dissolving therapeutic agents or helping to 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 such as urea. Stabilizers include: glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight peptides (e.g., peptides with 10 residues or fewer); 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; trisaccharides such as raffinose; and polysaccharides such as dextran. Stabilizers may be present in an amount of 0.5 to 10% by weight per weight of antigen-binding molecules.

[0211] 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.), poloxamers (184, 188, etc.), and pranic polyols. Nonionic surfactants can be present in the range of about 0.05 mg / mL to about 1.0 mg / mL (e.g., about 0.07 mg / mL to about 0.2 mg / mL).

[0212] Additional miscellaneous excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents.

[0213] 6.6.1. Pharmaceutical compositions for delivering nucleic acids encoding antigen-binding molecules

[0214] The antigen-binding molecules disclosed herein can be delivered via nucleic acids encoding antigen-binding molecules, such as plasmids, DNA, mRNA, or via viral vectors encoding antigen-binding molecules under the control of a suitable promoter.

[0215] Exemplary vectors include adenovirus- or AAV-based therapeutic agents. Non-limiting examples of adenovirus- or AAV-based therapeutic agents used in the methods, uses, or compositions described herein include, but are not limited to: rAd-p53, a recombinant adenovirus vector encoding the wild-type human tumor suppressor protein p53, for example, for the treatment of cancer (also known as Gendicine®, Genkaxin®, Qi et al., 2006, Modern Oncology, 14:1295-1297); Ad5_d11520, an adenovirus lacking the E1B gene for inactivating host p53 (also known as H101 or ONYX-015; see, for example, Russell et al., 2012, Nature Biotechnology 30:658-670); AD5-D24-GM-CSF, an adenovirus containing the cytokine GM-CSF, for example, for the treatment of cancer (Cerullo et al., 2010, Cancer). Res. 70:4297); rAd-HSVtk, a replication-deficient adenovirus possessing the HSV thymidine kinase gene, for example, for the treatment of cancer (developed as Cerepro®, ArkTherapeutics, see, for example, US Patent No. 6,579,855; developed by Advantagene as ProstAtak™; International PCT Application No. WO2005 / 049094); rAd-TNFα, a replication-deficient adenovirus vector expressing human tumor necrosis factor α (TNFα) under the control of a chemiradioactive EGR-1 promoter, for example, for the treatment of cancer (TNFerade™, GenVec; Rasmussen et al., 2002, Cancer Gene). Ther. 9:951-7; Ad-IFNβ, an adenovirus serotype 5 vector in which the E1 and E3 genes have been deleted, expresses the human interferon β gene under the guidance of the cytomegalovirus (CMV) immediate early promoter, for example for the treatment of cancer (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al., 2010, Mol. Ther. 18:852-860). Other vectors recognized in the art include, for example, lentiviral vectors (e.g., VSV), retroviral vectors, etc.

[0216] Any delivery vector now known or to be developed in the future, whether natural or engineered, can be used to deliver the antigen-binding molecules disclosed herein. In some embodiments, the delivery vector is a viral vector, for example, comprising a virus, a viral capsid, a viral genome, etc. In some embodiments, the delivery vector is a naked nucleic acid, for example, an episome. In some embodiments, the delivery vector comprises a nucleic acid complex. Exemplary non-limiting nucleic acid complexes used as delivery vectors include lipid complexes, polymeric vesicles, polymeric complexes, dendritic polymers, and inorganic nanoparticles (e.g., polynucleotide-coated gold, silica, iron oxide, calcium phosphate, etc.). In some embodiments, the delivery vectors described herein comprise a combination of a viral vector, a naked nucleic acid, and a nucleic acid complex.

[0217] In one embodiment, the delivery vector is a virus, including a retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus. In one embodiment, the delivery vector is adeno-associated virus (AAV), including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, or engineered or naturally selected variants thereof.

[0218] In one embodiment, the nucleic acid encoding the antigen-binding molecule (or a component thereof) also contains an adeno-associated virus (AAV) nucleic acid sequence. In one embodiment, the vector is a chimeric adeno-associated virus containing genetic elements from two or more serotypes. For example, an AAV vector having a rep gene from AAV1 and a cap gene from AAV2 (referred to as AAV1 / 2 or AAVRC1 / 2) can be used as a delivery vector to deliver nucleic acids expressing the antigen-binding molecule to cells or cells of patients in need.In one embodiment, the delivery carrier is AAV1 / 2, AAV1 / 3, AAV1 / 4, AAV1 / 5, AAV1 / 6, AAV1 / 7, AAV1 / 8, AAV1 / 9, AAV1 / 10, AAV1 / 11, AAV2 / 1, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2 / 10, AAV2 / 11, AAV3 / 1, AAV3 / 2, AAV3 / 4, AAV3 / 5, AAV3 / 6, AAV3 / 7, AAV3 / 8, AA V3 / 9, AAV3 / 10, AAV3 / 10, AAV4 / 1, AAV4 / 2, AAV4 / 3, AAV4 / 5, AAV4 / 6, AAV4 / 7, AAV4 / 8, AAV4 / 9, AAV4 / 10, AAV4 / 11, AAV5 / 1, AAV5 / 2, AAV5 / 3, AAV5 / 4, AAV5 / 6, AAV5 / 7, AAV5 / 8, AAV5 / 9, AAV5 / 10, AAV5 / 11, AAV6 / 1, AAV6 / 2, AAV6 / 3, AAV6 / 4, AAV6 / 5, AAV6 / 7, AA V6 / 8, AAV6 / 9, AAV6 / 10, AAV6 / 10, AAV7 / 1, AAV7 / 2, AAV7 / 3, AAV7 / 4, AAV7 / 5, AAV7 / 6, AAV7 / 8, AAV7 / 9, AAV7 / 10, AAV7 / 11, AAV8 / 1, AAV8 / 2, AAV8 / 3, AAV8 / 4, AAV8 / 5, AAV8 / 6, AAV8 / 7, AAV8 / 9, AAV8 / 10, AAV8 / 11, AAV9 / 1, AAV9 / 2, AAV9 / 3, AAV9 / 4, AAV9 / 5, AA V9 / 6, AAV9 / 7, AAV9 / 8, AAV9 / 10, AAV9 / 11, AAV10 / 1, AAV10 / 2, AAV10 / 3, AAV10 / 4, AAV10 / 5, AAV10 / 6, AAV10 / 7, AAV10 / 8, AAV10 / 9, AAV10 / 11, AAV11 / 1, AAV11 / 2, AAV11 / 3, AAV11 / 4, AAV11 / 5, AAV11 / 6, AAV11 / 7, AAV11 / 8, AAV11 / 9, AAV11 / 10, chimeric viral vectors or their derivatives.Gao et al., “Novel adeno-associated viruses fromrhesus monkeys as vectors for human gene therapy,” PNAS 99(18): 11854-11859, September 3, 2002, incorporated herein by reference, AAV vectors and chimeric viral vectors that can be used as delivery vectors, and their construction and uses.

[0219] 6.7. Generation Method

[0220] This disclosure includes methods for generating engineered protein hydrogenation (VH). A protein engineering method is disclosed, comprising generating engineered VH having an N-glycosylation site within 10 amino acids at its C-terminus. Methods for generating antigen-binding molecules comprising such engineered VHs are also disclosed.

[0221] In some embodiments, the disclosed method includes inserting an N-glycosylation site into the C-terminus of VH within 10 (e.g., 10, 9, 8, 7, 6, 5, 4, or 3) amino acids. In some embodiments, the method includes inserting an amino acid sequence X1X2X3X4X5NX6X7X8X9X 10 X 11 X 12 (SEQ ID NO:1) or a portion thereof is inserted into VH, wherein (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, wherein the amino acid is optionally not proline; (c) X7 is S or T, and (d) X8, X9, X 10 X 11 and X 12 Each amino acid is independently selected from: any amino acid and the absence of amino acids. In some embodiments, the method includes selecting the complete amino acid sequence X1X2X3X4X5NX6X7X8X9X 10 X 11 X 12 Insert into VH. In other embodiments, the method includes inserting the amino acid sequence X1X2X3X4X5NX6X7X8X9X... 10 X 11 X 12 A portion of the sequence is inserted into the VH, such that the resulting engineered VH contains the sequence NX6X7. For example, the sequence NX6X7 can be inserted into the C-terminus of the VH within 10 amino acids to produce an engineered VH.

[0222] In other embodiments, the disclosed method includes inserting, deleting, and / or substituting one or more amino acids within the sequence of VH, such that VH contains an N-glycosylation site within 10 (e.g., 10, 9, 8, 7, 6, 5, 4, or 3) amino acids at its C-terminus. In some embodiments, the method includes inserting, deleting, and / or substituting one or more amino acids within the sequence of VH, such that VH contains the amino acid sequence X1X2X3X4X5XNX6X7X8X9X within the 10 amino acids at its C-terminus. 10 X 11 X 12 (SEQ IDNO:1), where (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, wherein the amino acid is optionally not proline; (c) X7 is S or T, and (d) X8, X9, X 10 X 11 and X 12 Each amino acid is independently selected from: any amino acid and the absence of amino acids. The method may include inserting, deleting, and / or substituting 1, 2, 3, 4, 5, or more amino acids such that the VH contains an N-glycosylation site. In some embodiments, a single amino acid is inserted such that the engineered VH contains the sequence NX6X7, for example, by inserting an asparagine residue before the sequence SS or ST. In some embodiments, a single amino acid residue is substituted such that the engineered VH contains the sequence NX6X7, for example, by substituting an asparagine residue for X in the sequence XSS or XST to produce the sequence NSS or NST. In one embodiment, the method includes inserting an asparagine residue between the third and fourth positions of the sequence VTVSS (SEQ ID NO: 62) of the VH to produce the sequence VTVNSS (SEQ ID NO: 50) at the C-terminus of the engineered VH. In some embodiments, two or more amino acid residues are inserted, deleted, and / or substituted such that the engineered VH contains the sequence NX6X7, for example, by simultaneously inserting an asparagine residue before the sequence SX and also substituting X with a serine or threonine residue to produce the sequence NSS or NST. In one embodiment, the method includes inserting an N residue between the third and fourth positions of the sequence VTVSS (SEQ ID NO: 62) of VH, and also substituting a T residue at the fifth position of the sequence, thereby generating the sequence VTVNST (SEQ ID NO: 51) at the C-terminus of the engineered VH.

[0223] As disclosed herein, the engineered VH of this disclosure can be used to reduce the binding of antidrug antibodies to antigen-binding molecules. Therefore, methods for reducing the antigenicity of antigen-binding molecules are disclosed, comprising generating an antigen-binding molecule containing an engineered VH as disclosed herein. As used herein, “antigenicity” describes the degree to which an antigen-binding molecule binds to an antidrug antibody from a subject. In some embodiments, methods for reducing the antigenicity of an antigen-binding molecule by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more (e.g., as measured by an ADA reactivity assay as described in Section 9.1.5) are disclosed, comprising incorporating an N-glycosylation site into 10 amino acids at the C-terminus of one or more VHs of the antigen-binding molecule.

[0224] The terms "modification," "inserted amino acid," "deleted amino acid," and "substituted amino acid" used in this article are purely for convenience when referring to the generation of engineered VHs and do not require that the engineered VH be directly derived from or obtained from a specific VH. Such references describe engineered VHs that are sequence-related and have one or more sequence differences relative to a specific reference VH. For example, when the method for generating an engineered VH includes "substituting one or more amino acids of the VH," such descriptions include generating an engineered VH by mutagenesis of the coding sequence of the VH, and also include the direct synthesis of an engineered VH that has one or more amino acid differences relative to the VH (e.g., insertion, deletion, or substitution). Sometimes, for convenience only, a reference VH is referred to as a parental VH, and an antigen-binding molecule incorporated into the reference VH but not the engineered VH but otherwise identical is referred to as a corresponding antigen-binding molecule.

[0225] 6.8. Treatment methods

[0226] In some respects, this document discloses treatment methods comprising administering an antigen-binding molecule containing an engineered VH of the present disclosure. As described herein, engineered VH having an N-glycosylation site within 10 amino acids at its C-terminus, relative to the corresponding unengineered VH, exhibits reduced binding to antidrug antibodies. Therefore, without being bound by theory, the disclosed treatment methods involving the administration of an antigen-binding molecule containing an engineered VH of the present disclosure are understood to have reduced adverse immune responses and / or increased persistence relative to treatment methods involving the administration of a corresponding antigen-binding molecule in which the VH is not engineered.

[0227] In some embodiments, this disclosure provides a method for reducing adverse immune responses associated with antigen-binding molecule therapeutic agents. The method typically includes administering an antigen-binding molecule to a subject, the antigen-binding molecule comprising an engineered VH having an N-glycosylation site within 10 amino acids (e.g., 10, 9, 8, 7, 6, 5, 4, or 3 amino acids) at its C-terminus, such as the engineered VH described in Section 6.3. In specific embodiments, administration of an antigen-binding molecule comprising the engineered VH described herein reduces adverse immune responses associated with antigen-binding molecules (including unengineered VHs) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more. Following an adverse response to a corresponding antigen-binding molecule comprising an unengineered VH, an antigen-binding molecule comprising an engineered VH may be administered to the subject.

[0228] In some embodiments, this disclosure provides a method for increasing the therapeutic durability of an antigen-binding molecule therapeutic agent. The method typically includes administering an antigen-binding molecule to a subject, the antigen-binding molecule comprising an engineered VH having an N-glycosylation site within 10 amino acids (e.g., 10, 9, 8, 7, 6, 5, 4, or 3 amino acids) at its C-terminus, such as the engineered VH described in Section 6.3. In specific embodiments, administration of an antigen-binding molecule comprising the engineered VH described herein increases the therapeutic durability of the antigen-binding molecule (including the unengineered VH) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, or more. Following an adverse reaction to a corresponding antigen-binding molecule comprising the unengineered VH, an antigen-binding molecule comprising the engineered VH may be administered to the subject.

[0229] In some embodiments, certain antigen-binding molecules of this disclosure may be used to treat proliferative conditions (e.g., cancer) that express tumor-associated antigens. In specific embodiments, cancer includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt lymphoma, cancer of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, and so on. Tubal cancer, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, ocular cancer, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, glioma, head and neck cancer, hairy cell leukemia, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lobularia. Carcinoma, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck carcinoma with occult primary origin, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, papillomavirus. Paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary adenoma, pleural pulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Cezari syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoid tumor, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, or Wilms' tumor.

[0230] Table I below shows exemplary indications for the use of antigen-binding molecules that target specific TAAs.

[0231]

[0232] Other tumor-associated antigens and their corresponding indications are disclosed, for example, in Hafeez et al., 2020, Molecules25:4764, doi:10.3390 / molecules25204764, particularly in Table 1. Table 1 is incorporated herein by reference in its entirety.

[0233] 6.9. Combination Therapy

[0234] The antigen-binding molecules according to this disclosure can be administered in combination with one or more other pharmaceutical agents in a therapy. For example, the antigen-binding molecules of this disclosure can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any pharmaceutical agent administered to treat symptoms or diseases of a subject requiring such treatment. Such additional therapeutic agents may contain any active ingredient suitable for a particular indication being treated, preferably those having complementary activities that do not adversely affect each other. In some embodiments, the additional therapeutic agent is an immunomodulator, cell inhibitor, cell adhesion inhibitor, cytotoxic agent, apoptosis activator, or agent that increases the sensitivity of cells to apoptosis inducers. In specific embodiments, the additional therapeutic agent is an anticancer agent, such as a microtubule disruptor, antimetabolite, topoisomerase inhibitor, DNA intercalating agent, alkylating agent, hormone therapy, kinase inhibitor, receptor antagonist, tumor cell apoptosis activator, or antiangiogenic agent.

[0235] Other such agents are appropriately available in combinations of amounts effective for the intended purpose. The effective amount of these other agents depends on the amount of antigen-binding molecules used, the type of condition or treatment, and other factors discussed above. Antigen-binding molecules are typically used at the same dosage and route of administration as described herein, or approximately 1% to 99% of the dosage described herein, or at any dosage and route of administration as determined empirically / clinically.

[0236] Such combination therapies encompass combined administration (where two or more therapeutic agents are contained in the same or separate composition) and single administration, in which case the administration of the antigen-binding molecule of this disclosure may be performed before, simultaneously with, and / or after the administration of additional therapeutic agents and / or adjuvants.

[0237] 7. Sequence

[0238] Some sequences of this disclosure are provided in Table S below.

[0239] 8. Specific Implementation Examples

[0241] Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by way of examples with reference to the numbers set forth below.

[0242] 1. An antigen-binding molecule comprising a heavy chain variable domain (VH), wherein the VH contains an N-glycosylation site within 10 amino acids at the C-terminus of the VH.

[0243] 2. The antigen-binding molecule according to Example 1, wherein the N-glycosylation site is located within 8 amino acids at the C-terminus of the VH.

[0244] 3. The antigen-binding molecule according to Example 1, wherein the N-glycosylation site is located within 5 amino acids at the C-terminus of the VH.

[0245] 4. The antigen-binding molecule according to Example 1, wherein the N-glycosylation site is located within 4 amino acids at the C-terminus of the VH.

[0246] 5. The antigen-binding molecule according to Example 1, wherein the N-glycosylation site is located within 3 amino acids at the C-terminus of the VH.

[0247] 6. The antigen-binding molecule according to any one of Examples 1 to 5, wherein the N-glycosylation site is defined by the sequence motif NX[S / T], wherein X is any amino acid, optionally wherein the amino acid is not proline.

[0248] 7. An antigen-binding molecule comprising a heavy chain variable domain (VH), optionally wherein the antigen-binding molecule is an antigen-binding molecule according to any one of Examples 1 to 6, wherein the VH comprises the amino acid sequence X1X2X3X4X5XN6X7X8X9X at its C-terminus. 10 X 11 X 12 (SEQ ID NO:1), where (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, optionally wherein the amino acid is not proline; (c) X7 is S or T; and (d) X8, X9, X 10 X 11 and X 12 Each is independently selected from: any amino acid and none.

[0249] 8. The antigen-binding molecule according to any one of Examples 1 to 7, wherein X6 is S.

[0250] 9. The antigen-binding molecule according to any one of Examples 1 to 8, wherein X7 is S.

[0251] 10. The antigen-binding molecule according to any one of Examples 1 to 8, wherein X7 is T.

[0252] 11. The antigen-binding molecule according to any one of Examples 7 to 10, wherein X5 is V.

[0253] 12. The antigen-binding molecule according to any one of Examples 7 to 11, wherein X4 is T.

[0254] 13. The antigen-binding molecule according to any one of Examples 7 to 12, wherein X3 is V.

[0255] 14. The antigen-binding molecule according to any one of Examples 7 to 10, wherein X5 is S.

[0256] 15. The antigen-binding molecule according to any one of Examples 7 to 10 and 14, wherein X4 is S.

[0257] 16. The antigen-binding molecule according to any one of Examples 7 to 10 and 14 to 15, wherein X3 is V.

[0258] 17. The antigen-binding molecule according to any one of Examples 7 to 10 and 14 to 16, wherein X2 is T.

[0259] 18. The antigen-binding molecule according to any one of Examples 7 to 10 and 14 to 17, wherein X1 is V.

[0260] 19. The antigen-binding molecule according to any one of Examples 7 to 18, wherein X8 is P.

[0261] 20. The antigen-binding molecule according to any one of Examples 7 to 19, wherein X9 is P.

[0262] 21. The antigen-binding molecule according to any one of Examples 7 to 18, wherein X8 is K.

[0263] 22. The antigen-binding molecule according to any one of Examples 7 to 18 and 21, wherein X9 is P.

[0264] 23. The antigen-binding molecule according to any one of Examples 7 to 18, 21 and 22, wherein X 10 It is G.

[0265] 24. The antigen-binding molecule according to any one of Examples 7 to 18 and 21 to 23, wherein X 11 It is G.

[0266] 25. The antigen-binding molecule according to any one of Examples 7 to 18, wherein X8 is G.

[0267] 26. The antigen-binding molecule according to any one of Examples 7 to 18 and 25, wherein X9 is G.

[0268] 27. The antigen-binding molecule according to any one of Examples 7 to 18, 25 and 26, wherein X 10 It is G.

[0269] 28. The antigen-binding molecule according to any one of Examples 7 to 18 and 25 to 27, wherein X 11 It is G.

[0270] 29. The antigen-binding molecule according to any one of Examples 7 to 18, wherein X8, X9, X 10 X 11 and X 12 At least two of them do not exist.

[0271] 30. The antigen-binding molecule according to any one of Examples 7 to 18, wherein X8, X9, X 10 X 11 and X 12 At least three of them are missing.

[0272] 31. The antigen-binding molecule according to any one of Examples 7 to 18, wherein X8, X9, X 10 X 11 and X 12 At least four of them are missing.

[0273] 32. The antigen-binding molecule according to any one of Examples 7 to 18, wherein X8, X9, X 10 X 11 and X 12 None of them exist.

[0274] 33. The antigen-binding molecule according to any one of Examples 7 to 18, wherein the VH is at least 100 amino acids in length.

[0275] 34. The antigen-binding molecule according to any one of Examples 1 to 33, wherein the length of the VH is at most 125 amino acids.

[0276] 35. The antigen-binding molecule according to any one of Examples 1 to 34, comprising a single-chain Fv (scFv) containing the VH.

[0277] 36. The antigen-binding molecule according to Example 35, comprising a light chain variable domain (VL) located at the N-terminus of the VH.

[0278] 37. The antigen-binding molecule according to Example 35, comprising a light chain variable domain (VL) located at the C-terminus of the VH.

[0279] 38. The antigen-binding molecule according to any one of Examples 35 to 37, wherein the VH and VL are separated by a linker.

[0280] 39. The antigen-binding molecule according to any one of Examples 35 to 38, wherein the scFv is at least 225 amino acids in length.

[0281] 40. The antigen-binding molecule according to any one of Examples 35 to 39, wherein the length of the scFv is at most 300 amino acids.

[0282] 41. The antigen-binding molecule according to any one of Examples 1 to 34, further comprising VL.

[0283] 42. The antigen-binding molecule according to any one of Examples 1 to 34, which lacks VL.

[0284] 43. The antigen-binding molecule according to any one of Examples 1 to 42, wherein the antigen-binding molecule further comprises a dimerization moiety.

[0285] 44. The antigen-binding molecule according to Example 43, wherein the dimerization portion is an Fc domain.

[0286] 45. The antigen-binding molecule according to Example 44, wherein the Fc domain is located at the C-terminus of the VH.

[0287] 46. ​​The antigen-binding molecule according to Example 44, wherein the Fc domain is located at the N-terminus of the VH.

[0288] 47. The antigen-binding molecule according to Example 46, wherein the VH is located at the C-terminus of at least one polypeptide chain in the antigen-binding molecule.

[0289] 48. The antigen-binding molecule according to any one of Examples 45 to 47, wherein the Fc domain and the VH are separated by a linker.

[0290] 49. The antigen-binding molecule according to Example 48, wherein the linker is a glycine-serine linker.

[0291] 50. The antigen-binding molecule according to any one of Examples 1 to 49, further comprising an additional VH as defined in any one of Examples 1 to 34.

[0292] 51. The antigen-binding molecule according to Example 50, wherein the antigen-binding molecule is a dimer.

[0293] 52. The antigen-binding molecule according to Example 51, wherein the antigen-binding molecule is a homodimer.

[0294] 53. The antigen-binding molecule according to Example 51, wherein the antigen-binding molecule is a heterodimer.

[0295] 54. The antigen-binding molecule according to any one of Examples 1 to 53, wherein the VH is located at the N-glycosylation site and / or X1X2X3X4X5NX6X7X8X9X 10 X 11 X 12 The N residue of the (SEQ ID NO:1) sequence contains a glycan.

[0296] 55. The antigen-binding molecule according to Example 54, wherein the polysaccharide is G1F, G2F, G1F and GlcNAc, G2S, G2F and GlycNac or G2FS.

[0297] 56. The antigen-binding molecule according to any one of Examples 1 to 55, wherein the antigen-binding molecule is a multispecific antigen-binding molecule.

[0298] 57. An antigen-binding molecule, optionally an antigen-binding molecule according to any one of Examples 1 to 56, having Figure 1A The configuration shown.

[0299] 58. An antigen-binding molecule, optionally an antigen-binding molecule according to any one of Examples 1 to 56, having Figure 1B The configuration shown.

[0300] 59. An antigen-binding molecule, optionally an antigen-binding molecule according to any one of Examples 1 to 56, having Figure 1C The configuration shown.

[0301] 60. An antigen-binding molecule, optionally an antigen-binding molecule according to any one of Examples 1 to 56, comprising:

[0302] (a) a first polypeptide chain comprising, from its N-terminus to its C-terminus, (i) a first antigen-binding domain or a component thereof, (ii) a first dimerization moiety, and (iii) a second antigen-binding domain comprising VH as defined in any one of Examples 1 to 34; and

[0303] (b) A second polypeptide chain comprising, from the N-terminus to the C-terminus, (i) a third antigen-binding domain or a component thereof, and (ii) a second dimerization moiety, wherein the first dimerization moiety and the second dimerization moiety associate with each other to form the molecule.

[0304] 61. The antigen-binding molecule according to Example 60, wherein the second antigen-binding domain is scFv, optionally wherein the scFv is defined as in any one of Examples 35 to 40.

[0305] 62. The antigen-binding molecule according to Example 60 or 61, wherein the second polypeptide further comprises a fourth antigen-binding domain located at the C-terminus of the second dimerization moiety.

[0306] 63. The antigen-binding molecule according to Example 62, wherein the fourth antigen-binding domain comprises a VH, the VH comprising an N-glycosylation site within 10 amino acids at the C-terminus of the VH.

[0307] 64. The antigen-binding molecule according to Example 62 or 63, wherein the fourth antigen-binding domain is scFv.

[0308] 65. The antigen-binding molecule according to any one of Examples 62 to 64, wherein the first antigen-binding domain specifically binds to a T-cell antigen.

[0309] 66. The antigen-binding molecule according to any one of Examples 62 to 65, wherein the second antigen-binding domain specifically binds to a T-cell antigen.

[0310] 67. The antigen-binding molecule according to any one of Examples 62 to 66, wherein the third antigen-binding domain specifically binds to a T-cell antigen.

[0311] 68. The antigen-binding molecule according to any one of Examples 62 to 67, wherein the fourth antigen-binding domain specifically binds to a T-cell antigen.

[0312] 69. The antigen-binding molecule according to any one of Examples 65 to 68, wherein the T-cell antigen is CD3.

[0313] 70. The antigen-binding molecule according to any one of Examples 65 to 68, wherein the T-cell antigen is CD28.

[0314] 71. The antigen-binding molecule according to any one of Examples 62 to 66, wherein the third antigen-binding domain specifically binds to a tumor-associated antigen.

[0315] 72. The antigen-binding molecule according to any one of Examples 62 to 66, wherein the fourth antigen-binding domain specifically binds to a tumor-associated antigen.

[0316] 73. The antigen-binding molecule according to any one of Examples 60 to 72, wherein the first dimerization portion and the second dimerization portion are Fc domains.

[0317] 74. An antigen-binding molecule, optionally an antigen-binding molecule according to any one of Examples 1 to 56, comprising:

[0318] (a) First scFv;

[0319] (b) Connector; and

[0320] (c) Second scFv,

[0321] The first scFv or the second scFv includes VH as defined in any of Examples 1 to 34.

[0322] 75. The antigen-binding molecule according to Example 74, wherein the first scFv comprises a VH as defined in any one of Examples 1 to 34.

[0323] 76. The antigen-binding molecule according to Example 75, wherein the first scFv includes VL, a linker, and VH as defined in any one of Examples 1 to 34, from the N-terminus to the C-terminus.

[0324] 77. The antigen-binding molecule according to Example 75, wherein the first scFv includes VH, a linker, and VL as defined in any one of Examples 1 to 34, from the N-terminus to the C-terminus.

[0325] 78. The antigen-binding molecule according to any one of Examples 74 to 77, wherein the second scFv comprises VH as defined in any one of Examples 1 to 34.

[0326] 79. The antigen-binding molecule according to Example 78, wherein the second scFv includes VL, a linker, and VH as defined in any one of Examples 1 to 34 in the direction from the N-terminus to the C-terminus.

[0327] 80. The antigen-binding molecule according to Example 78, wherein the second scFv includes VH, a linker, and VL as defined in any one of Examples 1 to 34, from the N-terminus to the C-terminus.

[0328] 81. The antigen-binding molecule according to any one of Examples 1 to 80, wherein the antigen-binding molecule has reduced binding to anti-drug antibodies relative to the control antigen-binding molecule, for example, as measured by an ADA reactivity assay as presented in Section 7.1.5.

[0329] 82. The antigen-binding molecule according to any one of Examples 1 to 81, wherein the VH of the N-glycosylation site is a component of the T-cell binding antigen-binding domain (“TCE ABD”).

[0330] 83. The antigen-binding molecule according to Example 82, wherein the TCE ABD is capable of binding to components of the T cell receptor (TCR) complex.

[0331] 84. The antigen-binding molecule according to Example 83, wherein the component of the TCR complex is CD3.

[0332] 85. The antigen-binding molecule according to Example 83, wherein the component of the TCR complex is TCRαβ.

[0333] 86. The antigen-binding molecule according to Example 83, wherein the component of the TCR complex is TCRγδ.

[0334] 87. The antigen-binding molecule according to any one of Examples 82 to 86, wherein the TCE ABD (a) comprises the (i) CDR or (ii) VH and VL sequences of the antibody listed in Table T, or (b) competes with the antibody listed in Table T for binding to its target.

[0335] 88. The antigen-binding molecule according to any one of Examples 1 to 81, wherein the VH of the N-glycosylation site is a component of an antigen-binding domain (“TAA ABD”) capable of binding to tumor-associated antigens.

[0336] 89. The antigen-binding molecule according to Example 88, wherein the TAA ABD is capable of binding to any tumor-associated antigen identified in Section 6.4.2.

[0337] 90. The antigen-binding molecule according to Example 88, wherein the TAA ABD is capable of binding to the following: AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​proteins (e.g., GAGE-1 or -2), GD2, GD3, GloboH, phosphatidylinositol proteoglycan-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase or plaque protein-3.

[0338] 91. The antigen-binding molecule according to Example 88, wherein the TAA ABD is capable of binding to the following: CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, or CHK2.

[0339] 92. The antigen-binding molecule according to Example 88, wherein the TAA ABD is capable of binding to the following: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3.

[0340] 93. The antigen-binding molecule according to Example 88, wherein the TAA ABD is capable of binding to: multiligand proteoglycans, heparanases, integrins, osteopontin, connexins, cadherins, laminin, laminin-type EGF, lectins, fibronectin, notch, adhesion proteins (e.g., adhesion protein-4), tendinin, collagen (e.g., type X collagen), or matrix metalloproteinases.

[0341] 94. The antigen-binding molecule according to any one of Examples 1 to 81, wherein the antigen-binding molecule comprises (1) TCE ABD or a component thereof; and (2) TAA ABD or a component thereof.

[0342] 95. The antigen-binding molecule according to Example 94, wherein the VH containing the N-glycosylation site is a component of the TCE ABD.

[0343] 96. The antigen-binding molecule according to Example 95, wherein the TCE ABD is capable of binding to a component of the TCR complex.

[0344] 97. The antigen-binding molecule according to Example 96, wherein the component of the TCR complex is CD3.

[0345] 98. The antigen-binding molecule according to Example 96, wherein the component of the TCR complex is TCRαβ.

[0346] 99. The antigen-binding molecule according to Example 96, wherein the component of the TCR complex is TCRγδ.

[0347] 100. The antigen-binding molecule according to any one of Examples 95 to 99, wherein the TCE ABD (a) comprises (i) the CDR or (ii) VH and VL sequences of the antibody listed in Table T, or (b) competes with the antibody listed in Table T for binding to its target.

[0348] 101. The antigen-binding molecule according to Example 94, wherein the VH of the N-glycosylation site is a component of an antigen-binding domain (“TAA ABD”) capable of binding to tumor-associated antigens.

[0349] 102. The antigen-binding molecule according to Example 101, wherein the TAA ABD is capable of binding to any tumor-associated antigen identified in Section 6.4.2.

[0350] 103. The antigen-binding molecule according to Example 102, wherein the TAA ABD is capable of binding to the following: AFP, ALK, BAGE protein, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CALR, CEACAM5 (also known as carcinoembryonic antigen or CEA), CCR5, CD19, CD20 (MS4A1), CD22, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​proteins (e.g., GAGE-1 or -2), GD2, GD3, GloboH, phosphatidylinositol proteoglycan-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE protein, Ras, RGS5, Rho, SART-1, SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelle antigen (Tn), TRP-1, TRP-2, tyrosinase or plaque protein-3.

[0351] 104. The antigen-binding molecule according to Example 102, wherein the TAA ABD is capable of binding to the following: CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, or CHK2.

[0352] 105. The antigen-binding molecule according to Example 102, wherein the TAA ABD is capable of binding to the following: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or B7-H3.

[0353] 106. The antigen-binding molecule according to Example 102, wherein the TAA ABD is capable of binding to: multiligand proteoglycans, heparanases, integrins, osteopontin, connexins, cadherins, laminin, laminin-type EGF, lectins, fibronectin, notch, adhesion proteins (e.g., adhesion protein-4), tendinin, collagen (e.g., type X collagen), or matrix metalloproteinases.

[0354] 107. A pharmaceutical composition comprising an antigen-binding molecule according to any one of Examples 1 to 106.

[0355] 108. One or more nucleic acids encoding an antigen-binding molecule according to any one of Examples 1 to 106.

[0356] 109. A host cell engineered to express an antigen-binding molecule according to any one of Examples 1 to 106.

[0357] 110. A host cell transfected with one or more expression vectors, the expression vectors containing one or more nucleic acid sequences encoding an antigen-binding molecule according to any one of Examples 1 to 106 under the control of one or more promoters.

[0358] 111. A method for generating an antigen-binding molecule according to any one of Examples 1 to 106, the method comprising culturing a host cell according to Example 109 or 110 and recovering the antigen-binding molecule expressed therefrom.

[0359] 112. A method comprising administering to a subject an antigen-binding molecule according to any one of Examples 1 to 106.

[0360] 113. A method of treating cancer, comprising administering to a subject in need an antigen-binding molecule according to any one of Examples 1 to 106 or a pharmaceutical composition according to Example 107.

[0361] 114. The method according to Example 113, wherein the antigen-binding molecule is a bispecific antigen-binding molecule comprising TCE ABD and TAA ABD, and the cancer is associated with the expression of, for example, the tumor-associated antigen as shown in Table A.

[0362] 115. A polypeptide comprising an engineered VH as defined in any one of Examples 1 to 34.

[0363] 116. One or more nucleic acids encoding a polypeptide as described in Example 115.

[0364] 117. A host cell engineered to express the polypeptide described in Example 115 or the nucleic acid described in Example 116.

[0365] 118. A method for producing a polypeptide according to Example 115, the method comprising culturing a host cell according to Example 117.

[0366] 119. A protein engineering method comprising generating an antigen-binding molecule containing a VH having an N-glycosylation site within 10 amino acids at its C-terminus.

[0367] 120. A protein engineering method comprising generating a VH containing an N-glycosylation site within 10 amino acids at its C-terminus.

[0368] 121. A method for reducing the antigenicity of an antigen-binding molecule containing a VH, the method comprising incorporating an N-glycosylation site into 10 amino acids at the C-terminus of the VH.

[0369] 122. The method according to Example 121, wherein the antigen-binding molecule further comprises an additional VH.

[0370] 123. The method according to Example 122, wherein the method further comprises incorporating an N-glycosylation site into 10 amino acids at the C-terminus of the additional VH.

[0371] 124. The method according to any one of Examples 119 to 123, wherein the method comprises processing the amino acid sequence X1X2X3X4X5NX6X7X8X9X 10 X 11 X 12 (SEQ ID NO:1) or a portion thereof is inserted into the VH, wherein (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, optionally wherein the amino acid is not proline; (c) X7 is S or T, and (d) X8, X9, X 10 X 11 and X 12 Each is independently selected from: any amino acid and none.

[0372] 125. The method according to any one of Examples 119 to 123, wherein the method comprises substituting one or more amino acids within the sequence of the VH, such that the VH comprises the amino acid sequence X1X2X3X4X5XN6X7X8X9X within the 10 amino acids at its C-terminus. 10 X 11 X 12 (SEQ ID NO:1), where (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, optionally wherein the amino acid is not proline; (c) X7 is S or T, and (d) X8, X9, X 10 X 11 and X 12 Each is independently selected from: any amino acid and none.

[0373] 126. The method according to embodiment 124 or 125, wherein X6 is S.

[0374] 127. The method according to any one of Examples 124 to 126, wherein X7 is S.

[0375] 128. The method according to any one of Examples 124 to 126, wherein X7 is T.

[0376] 129. The method according to any one of Examples 124 to 128, wherein X5 is V.

[0377] 130. The method according to any one of Examples 124 to 129, wherein X4 is T.

[0378] 131. The method according to any one of Examples 124 to 130, wherein X3 is V.

[0379] 132. The method according to any one of Examples 124 to 128, wherein X5 is S.

[0380] 133. The method according to any one of Examples 124 to 128 and 132, wherein X4 is S.

[0381] 134. The method according to any one of Examples 124 to 128 and 132 to 133, wherein X3 is V.

[0382] 135. The method according to any one of Examples 124 to 128 and 132 to 134, wherein X2 is T.

[0383] 136. The method according to any one of Examples 124 to 128 and 132 to 135, wherein X1 is V.

[0384] 137. The method according to any one of Examples 124 to 136, wherein X8 is P.

[0385] 138. The method according to any one of Examples 124 to 137, wherein X9 is P.

[0386] 139. The method according to any one of Examples 124 to 136, wherein X8 is K.

[0387] 140. The method according to any one of Examples 124 to 136 and 139, wherein X9 is P.

[0388] 141. The method according to any one of Examples 124 to 136 and 139 to 140, wherein X 10 It is G.

[0389] 142. The method according to any one of Examples 124 to 136 and 139 to 141, wherein X 11 It is G.

[0390] 143. The method according to any one of Examples 124 to 136, wherein X8 is G.

[0391] 144. The method according to any one of Examples 124 to 136 and 143, wherein X9 is G.

[0392] 145. The method according to any one of Examples 124 to 136 and 143 to 144, wherein X 10 It is G.

[0393] 146. The method according to any one of Examples 124 to 136 and 143 to 145, wherein X 11 It is G.

[0394] 147. The method according to any one of Examples 124 to 136, wherein X8, X9, X 10 X 11 and X 12 At least two of them do not exist.

[0395] 148. The method according to any one of Examples 124 to 136, wherein X8, X9, X 10 X 11 and X 12 At least three of them are missing.

[0396] 149. The method according to any one of Examples 124 to 136, wherein X8, X9, X 10 X 11 and X 12 At least four of them are missing.

[0397] 150. The method according to any one of Examples 124 to 136, wherein X8, X9, X 10 X 11 and X 12 None of them exist.

[0398] 9. Example

[0399] 9.1. Materials and Methods

[0400] 9.1.1. Design and production of antigen-binding molecules

[0401] The antigen-binding molecule is designed to contain two peptides linked to each other via their Fc domains. The first peptide is designed to include, from the N-terminus to the C-terminus: a Fab targeting CD3, a first Fc domain, a linker, and a CD3-targeting scFv. The second peptide is designed to include, from the N-terminus to the C-terminus: a Fab targeting TAA, a second Fc domain, a linker, and a TAA-targeting scFv. The antigen-binding molecule is engineered by introducing amino acid modifications at or near the C-terminus of the scFv. Figure 2A The parent antigen-binding molecule is shown. Figures 2B to 2E Variants with C-terminal amino acid modifications that do not incorporate the N-glycosylation concordant sequence are shown, and Figure 2F and 2G A variant with an N-glycosylated concordant sequence incorporated at the C-terminus of scFv is shown.

[0402] All constructs were expressed in Expi293F™ cells via transient transfection (Thermo Fisher Scientific). Proteins in the Expi293F supernatant were purified using the ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) and a HiTrapMabSelect SuRe protein A column (GE Healthcare). After single-step elution with IgG elution buffer (Thermo Fisher Scientific), proteins were neutralized, dialyzed into a final buffer of 5% glycerol phosphate-buffered saline (PBS), aliquoted, and stored at -80°C.

[0403] The antigen-binding molecules and control constructs used in these examples are presented in Table 2.

[0404]

[0405] 9.1.2. Flow cytometry combined with assay

[0406] Cells were loaded at a rate of 1×10 6 Cells / mL were resuspended in FACS washing buffer (PBS containing 1% FBS and 0.5 mM EDTA). 1 × 10⁻⁶ cells / mL were then added to each well. 5 Individual cells were stained. Antibodies were applied at a 1:5 ratio from 1.3 x 10⁻⁶ cells. -07 The starting concentration of M was diluted. The diluted antibody was then added to wells containing cells. Cells were stained at 2°C to 8°C for 30 min and washed twice with FACS wash buffer. APC-conjugated goat anti-human IgG (Jackson Immuno Research, 109-607-003, 1:400) was added, along with a LIVE / DEAD fixed purple dead cell staining kit (Thermo Fisher Scientific), and cells were stained at 2°C to 8°C for 30 min. After washing, cells were fixed in 2% paraformaldehyde at 2°C to 8°C for 30 min. After two washes, stained cells were analyzed using a BD FACSCantoII instrument. Results were analyzed via FlowJo. FSC / SSC gating was used to select single cells, and BV421 negative gating was used to select live cells.

[0407] 9.1.3. Cytotoxicity assay

[0408] Tumor cell lines were resuspended in PBS and stained with CellTrace Purple (Thermo Fisher Scientific) according to the manufacturer's protocol. Cells were resuspended at a density of 100,000 cells / mL in RPMI medium (R10 medium) containing 10% FBS and penicillin-streptomycin-glutamine supplement, and 50 μL was seeded into 96-well plates to provide 5,000 cells per well. Peripheral blood mononuclear cells (PBMCs) were thawed and seeded at 1×10⁻⁶ cells / mL. 6 The PBMCs were resuspended in R10 medium at a density of 100 cells / mL and incubated overnight in a cell culture incubator. The next day, the PBMC suspension was mixed with human IgG1 at a final concentration of 5 mg / mL to simulate human serum, and the PBMC concentration was 2 × 10⁻⁶ cells / mL. 6Cells / mL. Add PBMCs to a final volume of 50,000 cells / well. Dilute the multispecific antigen-binding molecule 1:10 and add it to the assay plate to a final starting concentration of 6.7 x 10⁻⁶ cells / mL in R10 medium. -08 M. Incubate the plate in a cell culture incubator for 72 hours or longer. Then remove the supernatant and centrifuge at 300 xg for 4 minutes to pellet all cells in the suspension. Wash the tumor cells and harvest them with trypsin, then combine them with the suspended cell pellet. Wash the final cell pellet with PBS and stain with a LIVE / DEAD fixed near-infrared dead cell staining kit (Thermo Fisher Scientific). After washing twice with PBS, resuspend the cells in FACS wash buffer and analyze them on a cytometer such as the BD FACSCelesta or BD FACSCantoII. Results by FlowJo analysis: CellTrace-positive (BV421 channel) cells were gated to identify tumor cells, FSC / SSC gating was used to select single cells, and APC-Cy7 negative gating was used to select live cells. All appropriate compensating samples and fluorescence minus one (FMO) controls were included.

[0409] 9.1.4. Natural SEC-UV / MS

[0410] Intact mass measurements of mAbs were performed using native SEC-MS analysis to confirm that the molecular weight of the non-reduced mAb matched the predicted molecular weight of a given mAb based on the cDNA-derived amino acid sequence. Additionally, this method was able to identify and characterize any size variants that might be present in the sample (due to SEC separation). Native ESI mass spectra from each peak of the total ion chromatogram (TIC) from the SEC were averaged and deconvoluted using Intact Mass software from Protein Metrics, and the resulting mass spectra were output. Limited LysC digestion of the multispecific antigen-binding molecules REGN9930-VNST and REGN9930-VNSS yielded full-length scFv fragments (generated by preferential cleavage at the Lys site at the N-terminus of the GS linker). These fragments are well separated from mAbs or Fab and can therefore be distinguished from the N-ligands in the scFv region based on the predicted mass.

[0411] 9.1.5. ADA Reactivity Assay

[0412] To assess antigenicity, antibodies were tested in the ADA reactivity assay using electrochemiluminescence immunoassay (ECL) or the SMCxPro immunogenicity method to measure binding to the antidrug antibody. For the ECL method, the antibody was labeled with biotin or ruthenium. The labeled antibody was co-incubated with human or monkey serum. The bridged complex was captured by a streptavidin-coated plate, and the electrochemiluminescence signal generated by the activation of the ruthenium-labeled antibody by an electric current was recorded.

[0413] For the SMCxPro immunogenicity method, develop a kit according to the Millipore SMC immunogenicity plate-based assay protocol (catalog number 03-0189-00), labeling antibodies with biotin and Alexa647. Combine 1 μg / mL of biotinylated antibody, 0.063 μg / mL of Alexa647 antibody, and 1:10 diluted human or monkey serum in a 1:1:1 ratio and incubate overnight at 4°C with shaking. After incubation, transfer the bridged complex to a pre-blocked streptavidin-coated assay plate and incubate at room temperature with shaking for 1 hour. Wash the plate 6 times with 1X wash buffer. Add elution buffer B to the assay plate and incubate at room temperature with shaking for 10 minutes. After 10 minutes, quench the reaction with buffer C. Transfer the neutralized solution to an SMCxPro Aurora plate, seal, and read the value on the SMCxPro.

[0414] Samples with an average reaction rate of 2 times or higher than the monkey serum background were identified as pre-existing ADA positive. This background was determined individually for each antibody.

[0415] 9.2. Example 1: C-terminal modification does not affect the target binding affinity of REGN9930.

[0416] As described in Section 9.1.1, the antigen-binding molecule REGN9930 and its C-terminal modified constructs (Table 2) were generated. As described in Section 9.1.2, the binding affinity of the antigen-binding molecule to the target cell surface antigen was assessed by flow cytometry using Raji and Jurkat cells.

[0417] The binding affinity of the parental construct REGN9930 and the C-terminal engineered constructs to MAGE-A4 peptides overexpressed on Raji cells (MAGE-A4 (230-239) and MAGE-A4 (286-294)) and to CD3 expressed on Jurkat cells was evaluated. All constructs with C-terminal modifications showed binding affinity to Raji MAGE-A4 (230-239) cells, which was comparable to that of REGN9930. Figure 2AAll constructs with C-terminal modifications showed comparable binding affinity to Raji MAGE-A4 (286-294) cells to REGN9930. Figure 2B Similarly, all constructs exhibited binding affinity similar to that of Jurkat cells. These results indicate that the C-terminal modifications assessed in this instance did not affect the target binding affinity observed with the parental construct REGN9930.

[0418] 9.3. Example 2: C-terminal modification does not affect the cytotoxic efficacy of REGN9930.

[0419] The cytotoxic efficacy of the parental construct REGN9930 and the C-terminal engineered constructs was evaluated as described in Section 9.1.3. The percentage of A375 tumor cell viability was comparable to that of REGN9930 and all C-terminal engineered constructs (Figure 3), indicating that none of the C-terminal modifications affected the cytotoxic efficacy observed with the parental construct REGN9930.

[0420] 9.4. Example 3: Natural SEC-MS analysis of REGN9930-VNSS and REGN9930-VNST with limited LysC digestion

[0421] To determine whether the C-terminal modifications of the constructs REGN9930-VNSS and REGN9930-VNST had incorporated N-glycans, both constructs were subjected to limited LysC digestion and evaluated using natural SEC-UV / MS as described in Section 9.1.4.

[0422] Limited LysC digestion released the scFv domains at the Fc C-terminus of the two polypeptide chains attached to REGN9930-VNSS, generating a full-length scFv fragment. Although full-length mass spectrometry analysis revealed C-terminal modification, altering the C-terminal amino acid sequence VTVSS (SEQ ID NO: 62) to VTVNSS (SEQ ID NO: 50), no N-glycan occupancy at this site was detected. Figures 5A to 5E Similarly, when evaluating REGN9930-VNST, complete mass spectrometry analysis revealed the incorporation of a C-terminal modification, which altered the C-terminal amino acid sequence VTVSS (SEQ ID NO: 62) to VTVNST (SEQ ID NO: 51). However, as with REGN9930-VNSS, there was no evidence of N-glycan occupancy at the modified amino acid sequence. Figures 6A to 6D ).

[0423] 9.5. Example 4: C-terminal modification reduces the ADA reactivity of REGN9930

[0424] REGN9930 has pre-existing ADA reactivity. To assess the effect of C-terminal VH engineering on REGN9930 reactivity, ADA binding of six REGN C-terminal variants (Table 2) and REGN9930 and REGN8503 was evaluated using a subset of 24 treatment-naïve serum samples, as described in Section 9.1.5.

[0425] REGN8503 was used as a negative control and showed no pre-existing reactivity. In contrast, REGN9930 was associated with high levels of pre-existing reactivity. All C-terminal engineered variants of REGN9930 exhibited significantly reduced ADA binding (Figure 6). These results suggest that the C-terminal VH engineered variants of REGN9930 are effective in reducing the reactivity observed with REGN9930.

Claims

1. An antigen-binding molecule comprising a heavy chain variable domain (VH), wherein the VH contains an N-glycosylation site within 10 amino acids at the C-terminus of the VH.

2. The antigen-binding molecule according to claim 1, wherein the N-glycosylation site is located within 8 amino acids, 5 amino acids, 4 amino acids, or 3 amino acids at the C-terminus of the VH.

3. The antigen-binding molecule according to claim 1 or 2, wherein the N-glycosylation site is defined by the sequence motif NX[S / T], wherein X is any amino acid, optionally wherein the amino acid is not proline.

4. An antigen-binding molecule comprising a heavy chain variable domain (VH), optionally wherein the antigen-binding molecule is an antigen-binding molecule according to any one of claims 1 to 3, wherein the VH comprises the amino acid sequence X1X2X3X4X5XN6X7X8X9X at its C-terminus. 10 X 11 X 12 Wherein (a) X1, X2, X3, X4 and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, optionally wherein said amino acid is not proline; (c) X7 is S or T; and (d) X8, X9, X 10 X 11 and X 12 Each is independently selected from: any amino acid and none.

5. The antigen-binding molecule according to claim 4, wherein X6 is S.

6. The antigen-binding molecule according to claim 4 or claim 5, wherein X7 is S.

7. The antigen-binding molecule according to claim 4 or claim 5, wherein X7 is T.

8. The antigen-binding molecule according to any one of claims 4 to 7, wherein X5 is V.

9. The antigen-binding molecule according to any one of claims 4 to 8, wherein X4 is T.

10. The antigen-binding molecule according to any one of claims 4 to 9, wherein X3 is V.

11. The antigen-binding molecule according to any one of claims 4 to 7, wherein X5 is S.

12. The antigen-binding molecule according to any one of claims 4 to 7 and 11, wherein X4 is S.

13. The antigen-binding molecule according to any one of claims 4 to 7 and 11 to 12, wherein X3 is V.

14. The antigen-binding molecule according to any one of claims 4 to 7 and 11 to 13, wherein X2 is T.

15. The antigen-binding molecule according to any one of claims 4 to 7 and 11 to 14, wherein X1 is V.

16. The antigen-binding molecule according to any one of claims 4 to 15, wherein X8 is P.

17. The antigen-binding molecule according to any one of claims 4 to 16, wherein X9 is P.

18. The antigen-binding molecule according to any one of claims 4 to 15, wherein X8 is K.

19. The antigen-binding molecule according to any one of claims 4 to 15 and 18, wherein X9 is P.

20. The antigen-binding molecule according to any one of claims 4 to 15, 18 and 19, wherein X 10 It is G.

21. The antigen-binding molecule according to any one of claims 4 to 15 and 18 to 20, wherein X 11 It is G.

22. The antigen-binding molecule according to any one of claims 4 to 15, wherein X8 is G.

23. The antigen-binding molecule according to any one of claims 4 to 15 and 22, wherein X9 is G.

24. The antigen-binding molecule according to any one of claims 4 to 15, 22 and 23, wherein X 10 It is G.

25. The antigen-binding molecule according to any one of claims 4 to 15 and 22 to 24, wherein X 11 It is G.

26. The antigen-binding molecule according to any one of claims 4 to 15, wherein X8, X9, X 10 X 11 and X 12 At least two, at least three, at least four, or all of them are absent.

27. The antigen-binding molecule according to any one of claims 1 to 26, comprising a single-chain Fv (scFv) containing the VH.

28. The antigen-binding molecule according to any one of claims 1 to 27, wherein the antigen-binding molecule further comprises an Fc domain.

29. The antigen-binding molecule of claim 28, wherein the Fc domain is located at the N-terminus of the VH.

30. The antigen-binding molecule of claim 29, wherein the VH is located at the C-terminus of at least one polypeptide chain in the antigen-binding molecule.

31. The antigen-binding molecule according to any one of claims 1 to 30, wherein the VH is located at the N-glycosylation site and / or, when dependent on any one of claims 4 to 30, in the X1X2X3X4X5XN6X7X8X9X 10 X 11 X 12 The N residue of the sequence contains a glycan.

32. An antigen-binding molecule, optionally an antigen-binding molecule according to any one of claims 1 to 31, comprising: (a) a first polypeptide chain comprising, from its N-terminus to its C-terminus, (i) a first antigen-binding domain or a component thereof, (ii) a first dimerization moiety, and (iii) a second antigen-binding domain comprising VH as defined in any one of claims 1 to 26; and (b) A second polypeptide chain comprising, from its N-terminus to its C-terminus, (i) a third antigen-binding domain or a component thereof, and (ii) a second dimerization moiety, wherein the first dimerization moiety and the second dimerization moiety are associated with each other to form the molecule.

33. The antigen-binding molecule of claim 32, wherein the second antigen-binding domain is scFv, optionally wherein the scFv is as defined in claim 27.

34. An antigen-binding molecule, optionally an antigen-binding molecule according to any one of claims 1 to 31, comprising: (a) First scFv; (b) Connector; and (c) Second scFv, The first scFv or the second scFv includes VH as defined in any one of claims 1 to 26.

35. The antigen-binding molecule according to any one of claims 1 to 34, wherein the antigen-binding molecule has reduced binding to antidrug antibodies.

36. The antigen-binding molecule according to any one of claims 1 to 35, wherein the VH of the N-glycosylation site is a component of the T-cell binding antigen-binding domain ("TCE ABD").

37. The antigen-binding molecule according to any one of claims 1 to 35, wherein the VH of the N-glycosylation site is a component of an antigen-binding domain ("TAA ABD") capable of binding to tumor-associated antigens.

38. A pharmaceutical composition comprising an antigen-binding molecule according to any one of claims 1 to 37.

39. One or more nucleic acids encoding an antigen-binding molecule according to any one of claims 1 to 37.

40. A host cell transfected with one or more expression vectors, said expression vectors containing one or more nucleic acid sequences encoding an antigen-binding molecule according to any one of claims 1 to 37 under the control of one or more promoters.

41. A method for generating an antigen-binding molecule according to any one of claims 1 to 37, the method comprising culturing a host cell according to claim 40 and recovering the antigen-binding molecule expressed therefrom.

42. A method comprising administering to a subject an antigen-binding molecule according to any one of claims 1 to 37.

43. A method of treating cancer, comprising administering to a subject in need an antigen-binding molecule according to any one of claims 1 to 37 or a pharmaceutical composition according to claim 38, wherein the antigen-binding molecule is a bispecific antigen-binding molecule comprising TCE ABD and TAA ABD, and the cancer is associated with the expression of, for example, tumor-associated antigens as shown in Table A.

44. A polypeptide comprising an engineered VH as defined in any one of claims 1 to 37.

45. One or more nucleic acids encoding a polypeptide according to claim 44.

46. ​​A host cell engineered to express the polypeptide of claim 44 or the nucleic acid of claim 45.

47. A method for producing a polypeptide according to claim 44, the method comprising culturing a host cell according to claim 46.

48. A protein engineering method comprising generating a VH containing an N-glycosylation site within 10 amino acids at its C-terminus.

49. A method for reducing the antigenicity of an antigen-binding molecule containing VH, the method comprising incorporating an N-glycosylation site into 10 amino acids at the C-terminus of the VH.

50. The method of claim 49, wherein the method comprises substituting one or more amino acids within the sequence of the VH, such that the VH comprises the amino acid sequence X1X2X3X4X5XN6X7X8X9X within the 10 amino acids at its C-terminus. 10 X 11 X 12 Wherein (a) X1, X2, X3, X4, and X5 are each independently selected from any amino acid; (b) X6 is selected from any amino acid, optionally wherein said amino acid is not proline; (c) X7 is S or T, and (d) X8, X9, X 10 X 11 and X 12 Each amino acid is independently selected from: any amino acid and the absence thereof, optionally wherein the amino acid sequence X1X2X3X4X5NX6X7X8X9X is chosen. 10 X 11 X 12 As defined in any one of claims 5 to 26.

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