Fc region-based heterodimeric molecules and uses thereof

Abstract

The present invention relates to heterodimeric Fc scaffolds based on the Fc region of immunoglobulins, heteromultimeric proteins, in particular mono- or multispecific binding proteins, comprising said heterodimeric Fc scaffolds, and compositions and uses comprising the same.

Description

Fc region-based heterodimeric molecules and their uses Technical Field

[0001] The present invention relates to a heterodimeric Fc scaffold based on the immunoglobulin Fc region, a heteromultimeric protein comprising the heterodimeric Fc scaffold, in particular a single or multispecific binding protein, and compositions and uses thereof. Background Art

[0002] Bispecific antibodies are a new class of antibody drugs that have developed rapidly in recent years. These antibodies can simultaneously bind to two different antigens or two different epitopes on the same antigen, and therefore have the potential to bring additional clinical benefits to the treatment of complex diseases.

[0003] Currently, bispecific antibodies come in a variety of structural forms, including non-IgG-like formats based on the fusion of antibody fragments such as scFv, Fab, VH, and VL, and IgG-like formats based on the Fc domain. Compared to non-IgG-like formats, bispecific antibodies with conventional IgG structures can exhibit better physicochemical properties, stability, and pharmacokinetic characteristics, and have broad application prospects.

[0004] The Fc region of antibodies is known to have dimerization capabilities, playing a key role in maintaining antibody function in vivo. Various heterodimeric Fc technologies have been reported to assist in the production of bispecific antibodies (Moore GL, Methods 2019, 154:38–50).

[0005] For example, Carter et al. invented the knob-into-hole model and successfully achieved the preparation of bispecific antibodies (patent US7951917B1). In this method, a specific small side chain amino acid located at the interface of the CH3 region of the first heavy chain is mutated to a large side chain amino acid (e.g., T366Y), and a specific amino acid located at the interface of the CH3 region of the second heavy chain is mutated to a small side chain amino acid (e.g., Y407T), thereby causing the interface mutation residues to be complementary in spatial structure, thereby promoting the formation of Fc heterodimers. In addition to the above-mentioned example mutations, the more widely used mutation combinations in this method include the knob T366W and the hole T366S, L368A and Y407V mutation combinations, and the proportion of Fc heterodimers produced can reach 80-90%. However, the knob-into-hole model's ability to inhibit the formation of homodimer impurities is still insufficient.

[0006] Electrostatic steering has also been widely used to promote Fc heterodimer formation. This method mutates specific amino acids in the CH3 region of one heavy chain to positively charged amino acids (Lys or Arg) and specific amino acids in the CH3 region of the other heavy chain to negatively charged amino acids (Asp or Glu). This promotes Fc heterodimer formation through electrostatic attraction between the mutated residues. For example, Kannam et al. introduced mutations D399K and E356K in the CH3 region of the first heavy chain and mutations K409D and K392D in the CH3 region of the second heavy chain, generating a high proportion of heterodimers (patent US8592562B2). Igawa et al. also promoted Fc heterodimer formation by introducing mutations E356K and D399K in the CH3 region of the first heavy chain and mutations K439E and K409D in the CH3 region of the second heavy chain (patent US10011858B2).

[0007] Genmab invented DuoBody technology. This technology involves introducing an F405L mutation in the CH3 heavy chain of one antibody and a K409R mutation in the CH3 heavy chain of another antibody. These two antibodies are expressed and purified separately, then mixed in the presence of a reducing agent (such as 2-MEA) to achieve controlled Fab arm exchange between the two antibodies. This technology produces Fc heterodimers at a ratio of approximately 90-95% (patent US9150663B2).

[0008] Chain exchange kinase (SEED) is also used to promote Fc heterodimer formation (Muda et al. Proteins Eng. Des. Sel. 2011, 24: 447-454). This technique utilizes the sequence differences in the CH3 domains of IgA and IgG to generate Fc heterodimers by pairing complementary CH3 domains, thus avoiding homodimer formation.

[0009] Zymeworks has reported a scaffold structure that improves the stability of Fc heterodimers (patent US20120149876A1). For example, scaffold design 1a includes T366I, K392M, and T394W mutations in one heavy chain CH3 region and F405A and Y407A mutations in another heavy chain CH3 region, achieving a Tm value of 74°C. Furthermore, scaffold design 2a includes L351Y and Y047A mutations in one heavy chain CH3 region and T366V and K409F mutations in another heavy chain CH3 region, achieving a Tm value of 75.5°C. The proportion of Fc heterodimers based on this scaffold structure has reached over 90%.

[0010] Although various strategies for Fc heterodimer formation have been proposed, these strategies have exhibited certain drawbacks in their applications. For example, most Fc-containing bispecific antibodies suffer from homologous mismatching and reduced thermal stability, which affect the drugability of antibody drugs.

[0011] For example, it has been found that most Fc heterodimers formed using existing technologies have lower thermal stability than wild-type IgG1 monoclonal antibodies. The Tm value of the CH3 domain of wild-type IgG1 monoclonal antibodies is generally greater than 80°C (Ionescu RM, J Pharm Sci. 2008, 97:1414-1426). However, CH3 amino acid mutations introduced through Fc heterodimerization technology can significantly reduce the melting temperature (Tm) of the CH3 domain. For example, the Tm value of the CH3 heterodimer produced by the handle-hole model (T366W / T366S, L368A, Y407V) is 69.4°C (Atwell S, J. Mol. Biol. 1997, 270:26-35). The Tm value of the CH3 heterodimer produced using the electrostatic steering strategy (D399K, E356K / K409D, K392D) is 68.8°C (Gunasekaran K, J. Biol. Chem. 2010, 285:19637–19646). For structurally complex antibody drugs, a lower Tm value can affect drug manufacturing and storage. Although the introduction of non-native disulfide bonds (such as Y349C and S354C) in the handle-hole model can improve the efficiency of heteroFc dimer formation and thermal stability, for some drugs, such as antibody-drug conjugates (ADCs), the introduction of additional disulfide bonds can adversely affect product quality.

[0012] Furthermore, the proportion of Fc heterodimers produced by existing technologies generally ranges from 80-95%. Other impurities include Fc monomers, Fc homodimers, and high-molecular-weight aggregates. These impurities require further removal through ion exchange chromatography and other methods. In particular, the removal of homologous heavy chain mismatch impurities poses a challenge to downstream antibody purification processes. Furthermore, higher impurity levels lead to lower product recovery rates after purification, which in turn increases production costs.

[0013] Given the broad application prospects of IgG-like bispecific antibodies, there is still an urgent need in this field to develop Fc heterodimerization technology to solve the chain mispairing and thermal stability problems of bispecific antibodies, so as to improve the drugability of bispecific antibodies.

[0014] SUMMARY OF THE INVENTION

[0015] The inventors discovered that by introducing specific CH3 domain interface mutations in addition to the knob-into-hole (KIH) mutation into a scaffold structure based on a dimerized Fc region, antibodies containing this Fc scaffold exhibit superior thermal stability compared to corresponding antibodies containing only the KIH mutation. This approach also maintains the advantages of the KIH mutation (particularly, the advantage of promoting heavy chain pairing of the target heterodimer) and biological effects comparable to those of natural antibodies, including antigen-specific binding and binding to FcRn and FcγR receptors. Based on this discovery, the inventors proposed a combination of Fc heterodimerization mutations that can reduce the chain mispairing ratio and increase the stability of the target antibody product. The application of this combination of mutations can effectively improve the drugability and stability of bispecific antibodies.

[0016] In a first aspect, therefore, the present invention provides CH3 heterodimers and heterodimeric Fc scaffolds comprising combinations of mutations of the invention.

[0017] In a second aspect, the present invention provides the use of the heterodimeric Fc scaffold of the present invention and the CH3 heterodimer of the present invention as components for constructing heteromultimeric proteins, especially single / multi-specific binding proteins, and corresponding heteromultimeric proteins comprising the Fc scaffold or CH3 heterodimer.

[0018] In a third aspect, the present invention provides, inter alia, a binding protein comprising a heterodimeric Fc scaffold according to the present invention.

[0019] In a fourth aspect, the present invention provides isolated polynucleotides encoding heterodimeric Fc scaffolds, CH3 heterodimers, heteromultimeric proteins, or binding proteins according to the present invention; vectors (particularly expression vectors) comprising the isolated polynucleotides of the present invention; and host cells comprising the isolated polynucleotides or vectors (particularly expression vectors) of the present invention. The present invention also provides uses and methods of using the polynucleotides, vectors, or host cells of the present invention to produce heterodimeric Fc scaffolds, CH3 heterodimers, heteromultimeric proteins, and binding molecules of the present invention.

[0020] In a fifth aspect, the present invention provides a composition comprising a heteromultimeric protein or binding protein according to the present invention and a conjugate comprising a therapeutic agent conjugated to a heteromultimeric protein or binding protein according to the present invention. In some embodiments, the composition is a pharmaceutical composition comprising a heteromultimeric protein of the present invention (especially a multispecific binding protein of the present invention) and a pharmaceutically acceptable carrier.

[0021] In a sixth aspect, the present invention also provides methods of using and uses of the heteromultimeric proteins, binding proteins, compositions, and conjugates according to the present invention. In some embodiments, the present invention provides the use of the heteromultimeric proteins, binding proteins, compositions, or conjugates according to the present invention as a medicament. In some embodiments, the present invention provides the use of the heteromultimeric proteins, binding proteins, compositions, or conjugates according to the present invention for treating a disease in an individual in need thereof. In specific embodiments, the disease is cancer. The present invention also provides the use of the heteromultimeric proteins, binding proteins, compositions, or conjugates according to the present invention in the preparation of a medicament for treating a disease in an individual in need thereof; and a method of treating a disease in an individual using the heteromultimeric proteins, binding proteins, compositions, or conjugates according to the present invention, wherein the method comprises administering to the individual a therapeutically effective amount of the heteromultimeric proteins, binding proteins, compositions, or conjugates according to the present invention. In some embodiments, the heteromultimeric proteins, binding proteins, compositions, or conjugates are formulated in a pharmaceutically acceptable form. In some embodiments, the disease is cancer. In some embodiments, the individual is preferably a mammal, particularly a human.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 shows a schematic diagram of the partial crystal structure of the CH3-CH3 region of an Fc dimer.

[0024] FIG1A shows the hydrophobic interactions at the CH3-CH3 interface of wild-type IgG1 (PDB: 1HZH).

[0025] Figure 1B shows the hydrophobic interactions at the CH3-CH3 interface in the handle-hole model (PDB: 4NQS). The left side of the figure shows the handle chain, and the right side shows the hole chain.

[0026] Figure 1C shows the 3D structure of the CH3-CH3 region of the M2 mutation combination based on computer simulation. The hole L351Y mutation forms new hydrogen bonds with the handle T366W and E357 amino acids, respectively (indicated by the dotted lines).

[0027] Figure 1D shows the 3D structure of the CH3-CH3 region of the M3 mutation combination based on computer simulation. The handle K409D and hole D399R mutations form a new hydrogen bond network with multiple amino acids at the CH3-CH3 interface (indicated by the dotted lines).

[0028] FIG2 shows non-reducing SDS-PAGE electrophoresis of VHH-Fc / Fc mutant combinations.

[0029] In Figure 2A, the transfection ratio of the VHH-Fc and Fc plasmids was 1:1. In Figure 2B, the transfection ratio of the VHH-Fc and Fc plasmids was 0.6:1. For each mutant combination shown, the heterodimer (AB) and other impurities (including homodimers and free monomers) contained in the protein A purification product exhibited different migration distances due to differences in molecular weight.

[0030] FIG3 shows the Tm values ​​of VHH-Fc / Fc mutant combinations determined by DSC method.

[0031] The Tm values ​​of the samples were analyzed using a microcal PEAQ-DSC. Each mutation combination showed one to three Tm values ​​based on the Cp value, with the highest Tm value reflecting the stability of the CH3 domain.

[0032] FIG4 shows non-reducing SDS-PAGE electrophoresis of VHH-Fc / Fab-Fc mutant combinations.

[0033] For each mutation combination shown, the heterodimer (ABC) and other impurities (homodimer, half antibody, free monomer) contained in the protein A purification product exhibited different migration distances due to differences in molecular weight.

[0034] FIG5 shows the non-reducing SDS-PAGE electrophoresis of Fab-Fc mutants.

[0035] For each mutant shown, the heavy chain and light chain plasmid transfection ratio was 1:1.5. For each mutant shown, the protein A-purified product contained half-antibody monomers, disulfide-linked homodimers, and other impurities (free light chain and free heavy chain), which exhibited different migration distances due to differences in molecular weight.

[0036] FIG6 shows the in vitro assembly effect of Fab-Fc mutant combinations detected by HIC-HPLC.

[0037] Protein A affinity-purified half-antibody exhibits two main peaks, representing monomers or homodimers. Bispecific antibodies assembled in vitro exhibit a single main heterodimer peak, with other minor peaks representing half-antibody monomers or homodimer impurities.

[0038] FIG7 shows the binding activity of bispecific antibodies to antigens determined by ELISA.

[0039] FIG8 shows the binding affinity constants of bispecific antibodies to FcRn and FcγRI determined by Fortebio.

[0040] Detailed Description of the Invention

[0041] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0042] I. Definition

[0043] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0044] As used herein, the term "comprising" or "including" means including the stated elements, integers, or steps, but does not exclude any other elements, integers, or steps. For example, reference to an Fc region comprising a CH3 domain encompasses an Fc region consisting essentially of or consisting of a CH3 domain, as well as an Fc region further comprising other constant domains such as CH2.

[0045] As used herein, the term "Fc scaffold" refers to a scaffold protein dimer composed primarily of paired and dimerized immunoglobulin Fc regions. This dimer structure serves as a scaffold to which other functional molecules, including but not limited to binding domains, biologically active polypeptides or fragments thereof, and toxins, can be attached or conjugated at its N- or C-terminus. In some aspects, this scaffold can be used as a component for constructing multispecific (e.g., bispecific) antibodies or immunoadhesins.

[0046] As used herein, the term "heteromultimeric protein" refers to a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs from the first polypeptide in amino acid sequence by at least one amino acid residue. Preferably, the heteromultimeric protein is a monospecific or multispecific binding protein comprising at least one target binding domain. In some aspects, the first and second polypeptides of the heteromultimeric protein comprise a multimerization domain (e.g., the first and second CH3 domains of the CH3 heterodimer according to the present invention; or the first and second Fc regions of the heterodimeric Fc scaffold according to the present invention), wherein the multimerization domain promotes the first and second polypeptides to interact at the interface and form a "heterodimer". In some aspects, the heteromultimeric protein may comprise a "heterodimer" formed by the first and second polypeptides, or other polypeptides may be present in addition to the first and second polypeptides to form a higher order tertiary structure. Examples of heteromultimeric proteins include, but are not limited to, monospecific or multispecific binding proteins, such as bispecific antibodies, bispecific immunoadhesins, or antibody / immunoadhesin chimeras.

[0047] As used herein, the term "binding protein" is used interchangeably with "binding molecule" and is used in its broadest sense to refer to a protein molecule that specifically binds to at least one target. An example of a binding protein is an antibody and an immunoadhesin.

[0048] As used herein, the term "Fc scaffold-based binding protein" refers to a binding protein comprising an Fc scaffold and at least one target binding domain linked thereto, or consisting of or consisting essentially of an Fc scaffold and at least one target binding domain linked thereto. In some embodiments, the binding protein comprises one (and only one) Fc scaffold. In other embodiments, the binding protein may comprise more than one Fc scaffold.

[0049] As used herein, the term "monospecific" refers to a binding molecule that is capable of specifically binding to one and only one specific target (e.g., an epitope). The term "multispecific" refers to a binding molecule that is capable of specifically binding to at least two different targets (e.g., at least two different epitopes). Typically, a multispecific binding molecule comprises at least two target binding domains that are specific for different targets, respectively. In some embodiments, the different targets may be polypeptides or proteins or portions thereof, e.g., receptors, ligands, or antigenic epitopes, located on the same cell, different cells, the same antigen, or different antigens. In some embodiments, a multispecific binding molecule is bispecific and is capable of simultaneously binding to two different targets, e.g., two different epitopes, e.g., two different epitopes expressed on two different cells, or two different epitopes on two different antigens, or two different epitopes on one identical antigen.

[0050] As used herein, the term "valence" refers to the presence of a specific number of target binding domains in a binding molecule. Thus, the term "monovalent" in relation to a binding molecule refers to the presence of one (and no more than one) target-specific binding domain in the binding molecule. Accordingly, the term "multivalent" refers to the presence of multiple target-specific binding domains in the binding molecule. For example, a "tetravalent" binding molecule refers to a binding molecule in which a total of four target-specific binding domains are present, regardless of whether the targets targeted by the binding domains are the same.

[0051] As used herein, the term "target binding domain" refers to a portion of a polypeptide or protein that provides interaction with a target. Exemplary binding domains include the antigen binding domain of an antibody, a ligand, a receptor binding domain of a ligand, and a ligand binding domain of a receptor. In some aspects of the invention, a preferred target binding domain is the antigen binding domain of an antibody. The antigen binding domain of an antibody typically comprises amino acid residues from the complementary determining regions (CDRs) of the antibody. Natural immunoglobulin molecules typically have two antigen binding domains, and Fab molecules typically have a single antigen binding domain.

[0052] As used herein, the term "antigen binding domain" refers to an antibody or fragment thereof that specifically binds to an antigen epitope. In some embodiments, the antigen binding domain comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), or when the antibody is a heavy chain antibody, comprises a single heavy chain variable region (VHH). In some embodiments, the antigen binding domain may also comprise an antibody constant region. Available heavy chain constant regions include any of the following five types: α, δ, ε, γ, or μ. Available light chain constant regions include any of the following two types: κ and λ. Thus, in some aspects, a specific antigen binding domain may be selected from, including, but not limited to: antibody fragments comprising the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody, such as Fv, scFv, Fab, scFab, crossFab, and antibody fragments comprising the heavy chain variable region of a heavy chain antibody, such as VHH.

[0053] In this article, the term "antigenic determinant" is used interchangeably with "epitope", and refers to a site on macromolecules such as polypeptides and proteins that interacts with an antigen binding domain and forms a complex. In some aspects, an epitope can be, for example, a linear epitope consisting of a continuous amino acid segment or a conformational configuration consisting of discontinuous amino acid residues located in different regions. According to the present invention, useful antigenic determinants can be, for example, on tumor cell surfaces, virus-infected cell surfaces, other diseased cell surfaces, immune cell surfaces, or present in free macromolecules and / or extracellular matrix (ECM) in body fluids.

[0054] As used herein, the term "antigen" refers to a macromolecule that can elicit an immune response in a mammal, thereby producing specific antibodies against it. In some aspects, antigens useful according to the present invention are polypeptides or proteins, including, but not limited to, native forms of proteins and variants, fragments, or derivatives thereof from vertebrate sources. Vertebrates include mammals, such as primates, e.g., humans.

[0055] As used herein, the term "immunoglobulin" refers to a protein with the structure of a naturally occurring antibody. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 daltons consisting of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also known as a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3), also known as a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also known as a light chain variable domain, followed by a light chain constant domain (CL), also known as a light chain constant region. In an IgG antibody molecule, the VH-CH1 of the heavy chain is typically paired with the VL-CL of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG immunoglobulin essentially consists of two Fab molecules connected by an immunoglobulin hinge region and two dimerized Fc regions. The heavy chains of immunoglobulins can be assigned to one of five types based on the type of their constant region, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can also be assigned to one of two types, called κ and λ, based on the amino acid sequence of their constant domains.

[0056] As used herein, the term "antibody" refers to a polypeptide comprising one or more domains that bind to an epitope on an antigen of interest, wherein the binding domain has a variable region sequence derived from an immunoglobulin or has sequence identity thereto. The term encompasses various antibody structures, including but not limited to single-chain antibodies, single / multispecific antibodies, chimeric antibodies, humanized antibodies, human sequence antibodies, full-length antibodies, and antibody fragments, as well as antigen-binding proteins assembled therefrom as components, provided that they exhibit the desired antigen-binding activity. Typically, antibodies form an antigen-binding domain on the surface of a VH-VL dimer through three complementary determining regions (HCDR1-3) in their heavy chain variable region (VH) and three complementary determining regions (LCDR1-3) in their light chain variable region (VL). The six CDRs confer specific binding to the antibody and the antigen. However, in the case of heavy chain antibodies, such as heavy chain antibodies from Camelidae, the antibody can confer specific binding to the antigen through the three complementary determining regions (CDR1-3) in its single VH domain (also referred to herein as the VHH domain). The VHH domain, like the heavy and light chain variable regions of conventional IgG antibodies, contains four conserved framework regions (FRs) and three complementarity determining regions (CDRs), arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CD3-FR4.

[0057] As used herein, "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" refers to a region of an antibody variable region that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and / or contains antigen-contacting residues ("antigen contact points"). The CDRs are primarily responsible for binding to antigenic epitopes. In the VHH domain and VH / VL domain of the antibodies of the present invention, the CDRs are numbered sequentially from the N-terminus and are generally referred to as CDR1, CDR2, and CDR3. The CDR sequences in a defined VHH domain and a defined VH / VL domain can be determined using protocols well known in the art. Those skilled in the art can readily determine the CDR sequence ranges for any given antibody variable region amino acid sequence at http: / / www.abysis.org / abysis / , including the regional ranges of CDRs defined by the Kabat, AbM, Chothia, Contact, and IMGT schemes, and their combined ranges. Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways and combinations thereof, but preferably is a CDR sequence defined according to the Kabat scheme.

[0058] As used herein, the term "antibody fragment" refers to a non-complete portion of an antibody that contains an antigen-binding domain. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibodies, single-chain antibodies (e.g., scFv, scFab), and single-domain antibodies (sdAb). The amino acid sequence of VH or VL in an antibody fragment can be modified by substitution, deletion, addition, and / or insertion, as long as the antigen-binding ability is maintained. In addition, the variable regions of the antibody fragment can be chimerized and humanized.

[0059] In this article, "Fv domain" refers to the smallest antibody fragment that includes the complete antigen recognition site and binding site. "Fv" is a dimer (VH-VL dimer) formed by a VH and a VL strongly bound by non-covalent bonds.

[0060] As used herein, "scFv domain" refers to a single-chain polypeptide in which two variable regions (usually one VH and one VL) are connected via a linker to form the antigen-binding domain necessary for antigen binding.

[0061] As used herein, the term "Fab domain" refers to an antigen-binding domain similar to that formed by a heavy chain variable region VH and a heavy chain constant region CH1 (VH-CH1) pairing with a complementary light chain variable region VL and a light chain constant region CL (VL-CL) in a conventional four-chain IgG antibody.

[0062] As used herein, the term "crossFab" or "crossFab domain" refers to a Fab domain in which CH1 is exchanged with CL, ie, an antigen-binding domain formed by pairing VH-CL with VL-CH1.

[0063] As used herein, the term "scFab" refers to Fab domains connected into a single polypeptide chain via an artificial linker.

[0064] Herein, the term "VHH" or "VHH domain" is used to refer to the heavy chain variable domain of a heavy chain antibody lacking a light chain. Therefore, VHH is different from the conventional VH of a four-chain immunoglobulin in that it does not need to be paired with a light chain variable domain to form an antigen-binding domain. Such VHH molecules can be derived from antibodies produced in Camelidae species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides Camelidae can also produce heavy chain antibodies that naturally lack light chains, and such VHH are also within the scope of the present invention. In some cases, for the therapeutic application of VHH, it is desirable to reduce its immunogenicity. Therefore, preferably, the VHH domain used in the binding protein of the present invention comprises a humanized sequence.

[0065] As used herein, "immunoglobulin constant domain" refers to a constant domain from, obtained from, or derived from an immunoglobulin heavy chain (e.g., human IgG1 heavy chain) or light chain, including heavy chain constant domains CH1, CH2, CH3, and optionally CH4; and light chain constant domain CL. The term includes both native and variant sequence constant domains.

[0066] In this article, the "type" or "subtype" of an immunoglobulin constant domain refers to the type or subtype determined based on its amino acid sequence. The heavy chain constant domain can be classified into five different types according to its sequence: IgA, IgD, IgE, IgG and IgM, or further classified into a subtype (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Therefore, in this article, when referring to the IgG1 Fc region or CH3 domain, it means that the Fc region or CH3 domain can be classified as an IgG type based on its amino acid sequence, and can be further classified as an IgG1 subtype. Similarly, the light chain constant domain can be classified into kappa and lambda light chain CL domains according to its sequence. Those skilled in the art can easily determine the type or subtype to which it belongs by comparing the amino acid sequence of the constant domain with the sequence of the corresponding constant domain of natural immunoglobulins of different types or subtypes. In this context, when a heterodimeric Fc scaffold or heteromultimeric protein molecule of the present invention comprises multiple immunoglobulin heavy chain constant domains, it should be understood that they can be selected independently of each other based on the intended function or use of the molecule. As an example, for a heteromultimer of the present invention comprising a CH1 domain and CH2 and CH3 domains, all three constant domains can be of the IgG1 subtype, such as human IgG1, or only the CH3 domain or both the CH2 and CH3 domains can be of the IgG1 subtype, such as human IgG1.

[0067] As used herein, "IgG configuration" means that the antibody has the same configuration as an IgG immunoglobulin, essentially consisting of two Fab domains connected to the N-terminus of a dimerized Fc domain via an immunoglobulin hinge region (or, where appropriate, via a flexible linker peptide). Thus, in a typical case, an IgG-configured antibody consists of two heavy chains and two light chains, wherein each heavy chain has VH, CH1, CH2, and CH3 domains from N- to C-terminus; and each light chain has VL and CL domains from N- to C-terminus.

[0068] As used herein, "IgG-like configuration" means that the antibody still retains the same Y-shaped structural characteristics as IgG immunoglobulins, but one of the Fab domains is missing, and / or at least one Fab domain is replaced with a binding domain of a different configuration (e.g., scFv, VHH, ligand, or receptor ligand binding domain). Such IgG-like configuration antibodies can be described by indicating the type of binding domain connected to the Fc dimer scaffold. For example, VHH-Fc / Fc is a monovalent IgG-like configuration antibody composed of a VHH-Fc polypeptide and an Fc polypeptide; a VHH-Fc / Fab-Fc antibody is a bivalent IgG-like configuration antibody composed of a VHH-Fc polypeptide and a Fab-Fc polypeptide.

[0069] As used herein, the term "immunoadhesin" refers to an antibody-like protein molecule formed by fusing a non-immunoglobulin binding domain with the desired binding specificity (e.g., a binding domain from a cell surface receptor or ligand, or the ligand itself) with an immunoglobulin constant domain (e.g., an Fc region or CH3 domain). The immunoglobulin constant domain can be obtained from any immunoglobulin, such as IgG, particularly IgG1, IgG2, IgG3, or IgG4.

[0070] As used herein, the term "Fc domain" or "Fc region" is used to define the portion of the immunoglobulin heavy chain constant region that comprises the CH3 domain or a fragment thereof. In some cases, the immunoglobulin portion may further comprise one or more other immunoglobulin constant domains or fragments thereof, including a hinge region, a CH1 or CH2 domain. When referring to the amino acids of the Fc region and constant domains herein, the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991 is used. When referring to the human IgG1 Fc region, the EU numbering of amino acid residues can also be found in the IMGT Scientific chart (https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html), which is hereby incorporated by reference. According to this numbering, in the human IgG1 immunoglobulin heavy chain, amino acids 118-215 are the CH1 domain, amino acids 216-230 are the hinge region, amino acids 231-340 are the CH2 domain, and amino acids 341-447 are the CH3 domain. In some cases, the C-terminal lysine (Lys447) of the CH3 domain may not be present. Those skilled in the art can easily perform sequence alignments of published natural immunoglobulin constant regions to determine the constant domains, types / subtypes, and species origins contained in the heterodimeric Fc regions and binding proteins according to the present invention. In some embodiments, the first Fc region and the second Fc region of the heterodimeric Fc scaffold according to the present invention comprise or consist of a CH3 domain. In other embodiments, the Fc region further comprises a CH2 domain. In other embodiments, the Fc region further comprises a hinge region or a portion of a hinge region, for example, a portion of a lower hinge region comprising the sequence "CPPCP". As understood by those skilled in the art, the CH3 heterodimers of the present invention are a special form of the heterodimeric Fc scaffold according to the present invention, in which the first and second Fc regions of the Fc scaffold consist of, or consist essentially of, CH3 domains. Therefore, unless explicitly stated to the contrary, the description of the heterodimeric Fc scaffold of the present invention also applies equally to the CH3 heterodimers of the present invention.

[0071] In this article, the term "Fc domain" or "Fc region" covers native sequence Fc regions and variant sequence Fc regions. In this article, the term "native sequence Fc region" covers various naturally occurring immunoglobulin Fc region sequences, such as various Ig subtypes and their allotype Fc region sequences (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In this article, the term "variant sequence Fc region" refers to an Fc region polypeptide comprising a modification relative to a native sequence Fc region polypeptide. The modification can be the addition, deletion or substitution of amino acid residues. The substitution can include naturally occurring amino acids and non-naturally occurring amino acids. The purpose of the modification can be to change the physicochemical properties of the Fc region, such as thermal stability and heterodimerization tendency, and / or the binding of the Fc region to its receptor and its effector function.

[0072] Herein, an "Fc scaffold" is also referred to as an "Fc scaffold dimer" or a "dimeric Fc scaffold." Herein, the term "homodimeric" Fc scaffold refers to an Fc scaffold in which the first and second Fc regions comprising the scaffold are identical in sequence. Correspondingly, a "heterodimeric" Fc scaffold refers to an Fc scaffold in which the first and second Fc regions comprising the scaffold differ in sequence by at least one amino acid residue.

[0073] As used herein, the term "CH3 dimer" refers to a paired and dimerized pair of CH3 domains. The term "CH3 homodimer" means that the first and second CD3 domains comprising the dimer are identical in sequence. Correspondingly, a "CH3 heterodimer" means that the first and second CD3 domains comprising the dimer differ in sequence by at least one amino acid residue.

[0074] Herein, when a heterologous multimeric protein based on an Fc scaffold (e.g., a mono- / multi-specific binding protein) comprises only one Fc scaffold, such a multimeric protein can be considered as a dimer composed of two protein units (i.e., monomers) based on the two Fc regions of the scaffold, wherein one Fc member of the Fc scaffold and the polypeptide linked thereto (e.g., the target binding domain, which can be 0, 1, or more) constitute one protein unit (i.e., monomer) of the dimer; and the other Fc member of the Fc scaffold and the polypeptide linked thereto (e.g., the target binding domain, which can be 0, 1, or more) constitute the other protein unit (i.e., monomer) of the dimer. In this case, it should be understood that each monomer constituting the dimer can be a single or multiple polypeptide chains. When the two monomers are identical in sequence structure, that is, when they are copies of each other, the dimer is referred to herein as a homodimer. For example, natural IgG immunoglobulin is a typical example of such a homodimer. When the two monomers are different, for example, in the Fc sequence and / or the binding domain sequence to which they are attached, the dimer is referred to herein as a heterodimer. For example, bispecific IgG antibodies or IgG-like antibodies are typical examples of such heterodimers. Monospecific / multispecific antibodies based on heterodimeric Fc scaffolds are another typical example of such heterodimers.

[0075] As used herein, the term "purity" in connection with a heteromultimeric protein according to the present invention (e.g., a binding protein according to the present invention) refers to the proportion of the heteromultimeric protein relative to the total protein in the purified product after the heteromultimeric protein is expressed and purified from host cells. The purity of the target product can be determined by measuring the ratio of the target heteromultimeric protein product to various chain mismatch products in the purified product using SEC-HPLC. Preferably, the purity is greater than 90%.

[0076] As used herein, the term "thermal stability" in relation to a heteromultimeric protein according to the present invention (e.g., a binding protein according to the present invention) means that the protein exhibits a CH3 Tm value greater than 70°C, as determined by DSC; or, when the protein is an antibody of IgG configuration, exhibits a CH3 Tm value similar to that of a native human IgG1 immunoglobulin (e.g., ±2°C, preferably ±1°C). For heteromultimeric proteins according to the present invention based on a heterodimeric Fc scaffold, the Tm value is preferably determined without introducing non-native disulfide bonds into the heterodimeric Fc scaffold.

[0077] When referring to a single mutation, it is described by the amino acid residue position where the mutation occurs and the amino acid residues before and after the mutation, expressed as [original amino acid residue] mutated residue position [after mutation]. For example, the substitution of threonine at position 366 in the Fc region for tryptophan is expressed as T366W, and the substitution of tyrosine at position 409 in the Fc region for valine is expressed as Y407V. When referring to multiple mutation combinations, mutation combinations located on the same polypeptide chain are separated by a "-" symbol. For example, the combination of L351Y and D399R mutations occurring on the hole chain can be expressed as "L351Y-D399R." Mutation combinations located on different polypeptide chains are separated by a " / " symbol. For example, the combination of L351Y and D399R mutations occurring on the hole chain and K409D mutation occurring on the knob chain can be expressed as "hole L351Y-D399R / knob K409D."

[0078] As used herein, the terms "flexible connecting peptide" or "peptide linker" are used interchangeably to refer to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T), used alone or in combination, or from the hinge region of an immunoglobulin or a modified form thereof.

[0079] As used herein, the term "conjugate" refers to a binding protein modification or derivative formed by covalently linking or conjugating other molecules (e.g., therapeutic or diagnostic molecules) to a target protein (e.g., a heteromultimeric protein or binding protein according to the present invention). Examples of such other molecules include, but are not limited to, proteins / polypeptides / peptides, labels, drugs, and cytotoxic agents, such as radioisotopes; chemotherapeutic agents; growth inhibitors; enzymes and fragments thereof; fluorescent reporter proteins; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various known anti-tumor or anti-cancer agents.

[0080] As used herein, the "percentage (%) identity" of an amino acid sequence refers to the number of positions in the comparison window where the candidate sequence and the specific amino acid sequence set forth herein are aligned, and after introducing gaps, if necessary, to maximize the percentage identity, without considering any conservative substitutions as part of the sequence identity, at which the candidate sequence and the specific amino acid sequence have identical amino acid residues, and calculating the percentage of the total number of residues in the comparison window. Unless otherwise specified, the comparison window is the full length of the specific amino acid sequence.

[0081] As used herein, when referring to the constant domains CH2, CH3, and Fc domains, the term "wild type" means that the constant domain has a sequence derived from a natural immunoglobulin constant domain, or has no more than 1-5 amino acid residue changes compared to the natural sequence (preferably no more than 1, 2, 3, 4 or 5 amino acid residue changes, more preferably conservative amino acid substitutions).

[0082] With respect to polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following eight groups contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0083] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the polypeptide or protein molecule of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0084] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including, but not limited to, cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0085] As used herein, the term "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the subject is a human.

[0086] As used herein, the term "treatment" refers to clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

[0087] As used herein, the terms "cancer" and "tumor" are used interchangeably to refer to or describe the physiological condition in mammals that is generally characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and liquid tumors.

[0088] II. Heterodimeric Fc Scaffold

[0089] As demonstrated in the Examples, by introducing the specific Fc mutation combinations of the present invention, the heterodimeric Fc scaffold according to the present invention, after expression in cells and purification using an affinity chromatography column, can produce Fc heterodimers at a ratio exceeding 90%, a higher percentage than a control Fc protein containing only the corresponding knob-into-hole mutations. Further purification using an ion exchange column can achieve a purity exceeding 98%. Furthermore, compared to the control Fc protein, the purified Fc heterodimer exhibits improved thermal stability, with the CH3 domain Tm value exceeding 74°C as measured by DSC.

[0090] In a first aspect, the present invention provides a heterodimeric Fc scaffold that can be used to enhance the formation of desired heteromultimers. By using the heterodimeric scaffold according to the present invention, the yield of the desired heteromultimer product relative to unwanted heteromultimer and homomultimer impurities can be greatly increased, thereby improving the production efficiency and cost of the desired heteromultimer product; and simultaneously improving the thermal stability of the desired heteromultimer product.

[0091] The heterodimeric Fc scaffold according to the present invention comprises two paired and heterodimerized immunoglobulin Fc regions, wherein the two Fc regions each comprise a CH3 domain. The heterodimeric Fc scaffold according to the present invention is characterized in that the CH3 domain comprises not only a knob-into-hole (KIH) mutation but also the following mutations:

[0092] (a) the Fc region comprising a hole mutation further comprises an L351Y mutation; or

[0093] (b) The Fc region comprising the hole mutation further comprises L351Y and D339R mutations, and the Fc region comprising the knob mutation further comprises K409D mutation.

[0094] In some cases herein, for the sake of brevity, the above mutation combinations are also referred to as "CH3 interface characteristic mutations of the present invention." Similarly, for the sake of brevity, Fc regions with hole mutations and polypeptides comprising the same are also referred to as hole chains; and Fc regions with corresponding knob mutations and polypeptides comprising the same are referred to as knob chains. Accordingly, when referring to other mutations introduced into the hole chain, the term "hole" or "hole" is prefixed to clarify that the mutation occurs in the hole chain; similarly, when referring to other mutations introduced into the knob chain, the term "knob" or "knob" is prefixed to clarify that the mutation occurs in the knob chain. For example, hole L351Y refers to an L to Y residue substitution introduced into the hole chain Fc region at position L351; knob K409D refers to a K to D residue substitution introduced into the knob chain Fc region at position K409.

[0095] Knob-into-hole (KIH) mutation technology is known in the art. See, for example, US7951917B1; US ​​5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this mutation technology involves introducing a protrusion ("handle" or knob mutation) into the Fc region CH3 domain interface of a first polypeptide and a corresponding cavity ("hole" or hole mutation) into the Fc region CH3 domain interface of a second polypeptide, so that the protrusion can be positioned in the cavity to promote "heterodimer" formation of the first and second polypeptides and hinder homodimer formation. The protrusion can be constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acids (e.g., alanine or threonine), a complementary cavity identical or similar in size to the protrusion can be generated in the interface of the second polypeptide. Such combinations of protrusion and cavity mutations that complement each other in spatial positioning and size are referred to in the art as KIH mutations, wherein the protrusion mutation is referred to as a knob mutation of the KIH mutation and the cavity mutation is referred to as a hole mutation of the KIH mutation.

[0096] In a specific embodiment, an Fc scaffold according to the present invention comprises a KIH mutation, wherein the CH3 domain of one of the first and second Fc regions of the Fc scaffold comprises a knob mutation; and the CH3 domain of the other of the first and second Fc regions of the Fc scaffold comprises a complementary corresponding hole mutation. In a more specific embodiment, amino acid residue substitutions are introduced into the CH3 domain of one of the first and second Fc regions of the Fc scaffold according to the present invention (e.g., the first Fc region) such that one or more amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protrusion in the CH3 domain of that Fc region; and simultaneously, amino acid residue substitutions are introduced into the CH3 domain of the other of the first and second Fc regions of the Fc scaffold according to the present invention (e.g., the second Fc region) such that one or more amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of that Fc region that is sterically complementary to the protrusion, such that the protrusion present in the CH3 domain of one Fc region can be positioned within the cavity present in the CH3 domain of the other Fc region. The protrusions and cavities can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis, or by peptide synthesis. In some more specific embodiments, the Fc scaffold according to the present invention comprises a KIH mutation, wherein the introduced knob mutation is T366W and the introduced hole mutation is Y407V. In other more specific embodiments, the Fc scaffold according to the present invention comprises a KIH mutation, wherein the introduced knob mutation is T366W and the introduced hole mutation is T366S-L368A-Y407V.

[0097] In some embodiments, the Fc region of the heterodimeric Fc scaffold according to the invention consists of or consists essentially of a CH3 domain. In this embodiment, the invention thus provides a CH3 heterodimer according to the invention.

[0098] In some embodiments, in addition to the CH3 domain, the heterodimeric Fc scaffold according to the present invention or the heteromultimeric protein according to the present invention comprising the scaffold further comprises other immunoglobulin constant domains, for example, CH2 and / or hinge regions in the Fc region, and / or CH1 and / or CL domains connected to VH or VL in the target binding domain. In such embodiments, these immunoglobulin constant domains may be of the same or different species origin (but preferably all human origin), and / or of the same or different immunoglobulin types or subtypes (but preferably of the same IgG type or IgG subtype). In one embodiment, the Fc scaffold according to the present invention comprises CH2 and CH3 domains from an IgG (especially human IgG) immunoglobulin. In one embodiment, the Fc scaffold according to the present invention further comprises a hinge region and / or CH1 domain from an IgG (especially human IgG) immunoglobulin. As understood by those skilled in the art, when referring to the species origin and type of an immunoglobulin constant domain, it is meant that the constant domain comprises a native amino acid sequence of an immunoglobulin from the species and type or a variant sequence thereof, wherein the amino acid changes in the variant sequence do not affect the species origin and type or subtype determination of the constant domain. Typically, for the purposes of the present invention, the variant sequence has no more than 10 amino acid residue changes relative to the corresponding native sequence. As an example, the structural features of various IgG subtypes can be found in the review by Gestur Vidasson et al. (IgG Subclasses and Allotypes: From Structure to Effector Functions, Front Immunol. 2014; 5:520, doi:10.3389 / fimmu.2014.00520), which is incorporated herein by reference in its entirety. In some embodiments, the immunoglobulin constant domain contained in the heterodimeric Fc scaffold of the present invention or the heteromultimeric protein of the present invention is a constant domain of an IgG immunoglobulin. In some embodiments, the constant domains can be independently selected from different IgG1 subtypes, for example, IgG1, IgG2, IgG3 and IgG4 subtypes, but preferably they are all IgG1 subtype or IgG4 subtype. In some embodiments, the constant domains are human IgG immunoglobulin constant domains.

[0099] In some specific embodiments, the heterodimeric Fc scaffold according to the present invention comprises an IgG Fc region. In some more specific embodiments, the Fc region is an IgG1 Fc region. In other specific embodiments, the Fc region is an IgG4 Fc region.

[0100] In some embodiments, the Fc region of the heterodimeric Fc scaffold according to the present invention has an effector function. In some embodiments, the effector function is Fcγ receptor binding and / or FcRn receptor binding. In some embodiments, the Fcγ receptor is human FcγRIIa, FcγRI and / or FcγRIIIa, especially FcγRI. In some embodiments, compared to the native IgG1 dimer Fc domain (or a binding molecule comprising the native IgG1 dimer Fc domain), the heterodimeric Fc scaffold according to the present invention (or a binding molecule comprising the heterodimeric Fc scaffold) has an Fcγ receptor binding affinity of not less than 60%, for example not less than 50%, 40%, 30%, 20% or 10%. In some embodiments, the Fcγ receptor is FcγRI. In some embodiments, with respect to binding to the FcRn receptor, the heterodimeric Fc scaffold according to the present invention (or a binding molecule comprising the heterodimeric Fc scaffold) exhibits an FcRn binding affinity that is substantially similar to that of the native IgG1 dimer Fc domain (or a binding molecule comprising the native IgG1 dimer Fc domain), i.e., more than about 70% of the FcRn binding affinity of the native IgG1 dimer Fc domain, particularly more than about 80%, and more particularly more than about 90%.

[0101] The Fc domain can confer favorable pharmacokinetic properties to heteromultimeric proteins (e.g., bispecific antibodies) containing it, including extended serum half-life, good accumulation in target tissues, and favorable tissue-blood distribution ratios. However, the Fc domain may also cause heteromultimeric proteins (e.g., bispecific antibodies) containing it to target more cells expressing Fcγ receptors rather than the desired cells and / or tissues carrying the target of interest. In addition, activation of the Fcγ receptor signaling pathway may lead to cytokine release, causing heteromultimeric proteins containing Fc domains to cause serious side effects after systemic administration. In some embodiments, in addition to the characteristic mutations of the CH3 interface of the present invention, the heterodimeric Fc scaffold of the present invention may contain or not contain other mutations according to specific needs to maintain or affect its (one or more) effector functions. For example, it is known that the upper CH2 domain and hinge region of antibodies are involved in various effector functions, such as Fcγ receptor binding and effector functions such as ADCC; some CH2 and CH3 interface residues of antibodies are involved in FcRn receptor binding. In some cases, the Fc region used in the heterodimer scaffold of the present invention can be modified (reduced or enhanced) by including additional mutations introduced into the CH2 domain and / or hinge region (if present) to alter (reduce or enhance) one or more desired effector functions, for example, the binding affinity of the Fc region to Fcγ receptors and / or FcRn. Such amino acid modifications are known in the art and include, but are not limited to, amino acid substitutions at one or more positions selected from E233, L234, L235, N297, P331, and P329, more specifically, one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, and P331S. In some cases where the Fc scaffold has a human IgG1 Fc region, binding of the Fc scaffold to Fcγ receptors can be substantially reduced by including a L234A-L235A mutation, a L234A-L235A-P329G mutation, and / or a N297A mutation in the Fc region.

[0102] In the present invention, some exemplary sequences of Fc scaffolds according to the present invention are provided. In some embodiments, the heterodimeric Fc scaffold of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first Fc region and the second polypeptide comprises a second Fc region, wherein:

[0103] (a) the CH3 domains of the first and second Fc regions, in addition to the characteristic CH3 interface mutations of the present invention, each further comprise 0-3 amino acid residue changes relative to a wild-type CH3 domain. Preferably, the wild-type CH3 domain is a native IgG (especially IgG1 or IgG4, preferably human IgG1) immunoglobulin CH3 domain, more preferably comprising the amino acid sequence of SEQ ID NO: 19 or 20;

[0104] (b) the first and second Fc regions comprise a native IgG (especially IgG1 or IgG4, preferably human IgG1) immunoglobulin CH2 domain, or a CH2 domain comprising the amino acid sequence of SEQ ID NO: 21 or having 1-5 amino acid residue changes therefrom;

[0105] (c) the Fc region comprising a knob mutation comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto;

[0106] (d) the Fc region comprising a hole mutation comprises the amino acid sequence of SEQ ID NO: 7 or 8, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical thereto, and / or

[0107] (e) The Fc scaffold does not form a non-native disulfide bond between the first Fc region and the second Fc region.

[0108] In some preferred embodiments, the Fc region comprising a knob mutation comprises a CH3 domain having an amino acid sequence as set forth in SEQ ID NO: 1 or 2, or an amino acid sequence having at least 95% identity thereto; and the Fc region comprising a hole mutation comprises a CH3 domain having an amino acid sequence as set forth in SEQ ID NO: 7 or 8, or an amino acid sequence having at least 95% identity thereto. In some embodiments, the Fc region with the knob mutation comprises a CH3 domain having an amino acid sequence as set forth in SEQ ID NO: 1 or 2, and the Fc region with the hole mutation comprises a CH3 domain having an amino acid sequence as set forth in SEQ ID NO: 7 or 8. In some embodiments, the Fc region with the knob mutation comprises a CH3 domain having an amino acid sequence as set forth in SEQ ID NO: 1, and the Fc region with the hole mutation comprises a CH3 domain having an amino acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the Fc region with the knob mutation comprises a CH3 domain having an amino acid sequence as set forth in SEQ ID NO: 2, and the Fc region with the hole mutation comprises a CH3 domain having an amino acid sequence as set forth in SEQ ID NO: 8. In any of the above embodiments, the Fc region comprising the knob mutation can be the first Fc region and the Fc region comprising the hole mutation is the second Fc region, or vice versa.

[0109] In some embodiments, the heterodimeric Fc scaffold according to the present invention does not contain non-native disulfide bonds introduced into the interface of the first and second Fc region CH3 domains, and still has good thermal stability, for example, the Tm value of the CH3 domain is 70°C or higher as determined by DSC.

[0110] In some embodiments, the heterodimeric Fc scaffold according to the present invention has better thermal stability than a corresponding control Fc scaffold with the same KIH mutation. As used herein, a "corresponding control Fc scaffold" refers to a heterodimeric Fc scaffold that is identical in sequence structure (including the KIH mutation) to the heterodimeric Fc scaffold of the present invention with the exception of the unmutated native residues at positions L351, D366, and K409.

[0111] In some embodiments, the heterodimeric Fc scaffolds according to the present invention have comparable purity compared to a corresponding control Fc scaffold having the same KIH mutation. In some embodiments, after expression and assembly from host cells and purification under the same conditions, the purity of the heterodimeric Fc scaffolds according to the present invention is at least 90%, 95%, 100%, 110% or more of the purity of the control Fc scaffold, and preferably has a purity greater than 90% as determined by SEC-HPLC.

[0112] III. Heteromultimeric proteins

[0113] In a second aspect, the present invention provides a heteromultimeric protein comprising a CH3 heterodimer according to the present invention or a heterodimeric Fc scaffold according to the present invention.

[0114] In some embodiments, a heteromultimeric protein according to the present invention comprises at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise a first and a second CH3 domain, respectively, wherein the first and second CH3 domains in the first and second polypeptides meet at an interface and form a CH3 heterodimer according to the present invention.

[0115] In some embodiments, a heteromultimeric protein according to the present invention comprises at least a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprise a first and a second Fc region, respectively, wherein the first and second Fc regions in the first and second polypeptides meet at an interface and form an Fc scaffold according to the present invention.

[0116] In some embodiments, the heteromultimeric protein is a single or multispecific binding protein comprising at least one target binding domain, wherein the at least one target binding domain is connected to a CH3 heterodimer according to the present invention or a heterodimeric Fc scaffold according to the present invention. In some embodiments, the heteromultimeric protein according to the present invention comprises more than one Fc scaffold. In some embodiments, the heteromultimeric protein according to the present invention comprises only one Fc scaffold. Herein, such single or multispecific binding proteins comprising the Fc scaffold of the present invention are also referred to as "Fc scaffold-based binding proteins."

[0117] Fc scaffold-based binding proteins

[0118] In some aspects, therefore, the present invention provides binding proteins based on the heterodimeric Fc scaffold of the present invention.In some embodiments, the binding protein according to the present invention comprises at least one target binding domain linked to the Fc scaffold.

[0119] Type of target binding domain

[0120] There is no particular limitation on the type of target binding domain that can be used in the binding proteins of the present invention, as long as it has the ability to bind to the target of interest. Such binding domains, as known in the art, can be from binding partners, including non-immunoglobulin-based binding pairs, such as receptor / pairing binding pairs, enzyme / substrate pairs, or immunoglobulin-based binding pairs, such as antigen / antibody binding pairs, antibody / anti-idiotypic antibodies. When the target binding domain is from an immunoglobulin-based binding pair, the binding protein comprising it can be an antibody, such as a single / multi-specific antibody. When the target binding domain is from a non-immunoglobulin-based binding pair, the binding protein comprising it can be an immunoadhesin. In addition, the present invention also contemplates binding proteins comprising binding domains from both of these two different binding pairs, such antibody / immunoadhesin chimeric proteins sometimes also referred to herein as antibodies.

[0121] In some embodiments, the binding protein according to the present invention comprises at least one antigen binding domain. Antigens that can be bound to the antigen binding domain according to the present invention can be selected from, for example, but not limited to: tumor-associated antigens, immune checkpoint molecules, angiogenic factors, or combinations thereof. Tumor-associated antigens may include, but are not limited to: MUC1-1, BCMA, CLDN18.2, HER2, BRAF, EGFR, CD20, CD38, FolR1, and CD52. Immune checkpoint molecules may include, but are not limited to: PD-L1, PD-1, PD-L2, CTLA-4, B7-H3, TIM3, LAG-3, VISTA, ICOS, 4-1BB, OX40, GITR, and CD40. Angiogenic factors may include, but are not limited to, basic FGF, HGF, Stie-2, Svegfr-1, Svegfr-2, EGF, IL-6, IL-8, PLGF, VEGF, PDGF-bb, ANG1, ANG2, SDF-1α, MDC, Galectin, TSP-1, Endocan, Enos, HIF-1α. In some embodiments, the antigen is selected from tumor-associated antigens, for example, EGFR and HER2.

[0122] In some embodiments, binding proteins according to the invention comprise at least one non-immunoglobulin binding domain.

[0123] In some embodiments, examples of such non-immunoglobulin binding domains that can be used include, but are not limited to, ligand binding domains from the following receptor proteins: 4-1BB; adrenocorticotropic hormone receptor; activin receptor; BLTR (leukotriene B4 receptor); BMP receptor; C3a receptor; C5a receptor; chemokine receptor; cytokine receptor; growth hormone receptor; BTLA; interferon-α receptor; interferon-β receptor; interferon-γ receptor; type I IL-1 receptor; type II IL-1 receptor; IL-10 receptor; IL-11 receptor; IL-12 receptor; BCMA; TACI; BAFF receptor; immunomodulatory signaling receptor CD72; Kaposi's sarcoma-associated herpes virus GPCR; lipoxin A4 receptor; lymphotoxin beta receptor; RON receptor; SCF receptor; somatostatin receptor; T1 / ST2; TGF-β receptor; tumor necrosis factor receptor; TNFRSF19; erythropoietin receptor; leukemia inhibitory factor receptor; and C-kit receptor.

[0124] In some embodiments, examples of such non-immunoglobulin binding domains that can be used include, but are not limited to, receptor binding domains from the following ligands or the ligands themselves: α-MSH; 9E3 / cCAF; adrenocorticotropic hormone; activin; AK155; angiogenesis inhibitors; Apo2L / TRAIL; BLR1 ligand / BCA-1 / BLC / CXCL13; calcitonin gene-related peptide; CD27 ligand; CD30 ligand; CD40 ligand; endorphin; endostatin; erythropoietin; Fas ligand; Flt-3 ligand; G-CSF; GCP- 2 / CXCL6; GM-CSF; various cytokines, such as IFNα, IFNβ; interferon gamma; IL-1α; IL-1β; IL-10; IL-11; IL-12; IL-13; IL-15; IL-16; IL-2; IL-27; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8 / CXCL8; IL-9; somatostatin; stem cell factor; substance P; TARC / CCL17; TCA3 / mouse CCL1; TECK / CCL25; TGFβ; thrombopoietin; TNFα.

[0125] In some embodiments, the non-immunoglobulin binding domain is from a protein selected from the following: VEGF receptor; TNF receptor; IL-1; lymphocyte function-associated antigen 3 (LFA-3 / CD58); lymphotoxin beta receptor (LTBR); CTLA-4; IL-12; activin receptor; TACI; BR3; T cell receptor; CD4; L-selectin; homing receptor; CD44; NP receptor; interferon gamma receptor; 4-1BB and IgE receptor. In other embodiments, the non-immunoglobulin binding domain is from a receptor binding domain of a ligand selected from the following: IL-1, IL-12. Examples of other available non-immunoglobulin binding domains can also be found in, for example, US7951917B1 and US20220275048A1.

[0126] Target binding domain connection mode

[0127] In the binding protein according to the present invention, the connection position of the target binding domain on the Fc scaffold is not particularly limited. In some embodiments, the at least one target binding domain is independently connected to the N-terminus or C-terminus of the first and / or second polypeptide of the Fc scaffold. In some embodiments, at least two or more or all of the at least one target binding domain are optionally connected to each other via a peptide linker, and then connected to the heterodimeric Fc scaffold according to the present invention. In other embodiments, at least two or more or all of the at least one target binding domain are optionally connected to different ends of the Fc scaffold via a peptide linker. For example, when the binding protein comprises two binding domains, one binding domain can be connected to the N-terminus of the first polypeptide of the Fc scaffold, while the other binding domain is connected to different ends of the Fc scaffold, for example, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, or the C-terminus of the second polypeptide. Therefore, in the binding protein according to the present invention, at least one, two, three or all of the four ends of the heterodimeric Fc scaffold (i.e., the N-terminus and C-terminus of the first and second polypeptides) can each be connected to at least one (preferably 1 or two) target binding domains.

[0128] The at least one target binding domain contained in the binding protein can be identical or different from each other. In one embodiment, at least two, more, or all of the at least one target binding domain are identical. In other embodiments, at least two, more, or all of the at least one target binding domain are different from each other.

[0129] In some embodiments, the binding protein is monospecific and has a single or multiple binding domains for that specificity. In some embodiments, the binding protein is multispecific and preferably has a single or multiple binding domains for each specificity.

[0130] In some embodiments, the at least one target binding domain is independently an antigen binding domain selected from Fab, VHH, scFv, scFab and crossFab. In the case where the binding domain is Fab or crossFab, the Fab or crossFab can be connected to another binding domain or Fc scaffold of the binding protein through the C-terminus of the chain comprising the VH domain or the C-terminus of the chain comprising the VL domain, optionally via a peptide linker, for example, to the N-terminus of the first or second polypeptide of the heterodimeric Fc scaffold according to the present invention.

[0131] In some embodiments, at least one of the at least one target binding domain is a VHH domain. In other embodiments, at least one of the at least one target binding domain is a Fab domain. In other embodiments, at least one of the at least one target binding domain is a VHH domain and at least one of the at least one target binding domain is a Fab domain. In other embodiments, at least two, more than one, or all of the at least one target binding domain are Fab domains, and the Fab domains have the same or different VLCL light chains.

[0132] In some embodiments, the binding protein is a monospecific monovalent protein comprising a VHH domain, and preferably the VHH domain is connected to the N-terminus of the first or second polypeptide of the Fc scaffold. In some embodiments, the binding protein is a bispecific bivalent binding protein comprising a VHH and / or Fab binding domain. In some embodiments, the binding protein is a bispecific antibody of IgG configuration comprising a first and a second Fab domain and an Fc scaffold, preferably, the first and second Fab domains are connected to the N-terminus of the first and second polypeptides of the Fc scaffold respectively through the C-terminus of the chain comprising the VH domain. In some embodiments, the binding protein is a bispecific antibody of IgG-like configuration comprising VHH and Fab, wherein the antibody consists of a heavy chain comprising a VHH-Fc domain, a heavy chain comprising a VH-Fc domain, and a light chain comprising a VL-CL domain.

[0133] In this article, the term "connection" used when referring to different components in a scaffold or binding protein refers to the fusion or conjugation of the components directly or via a suitable linker. The peptide linker commonly used for this purpose is a short amino acid polypeptide with a certain flexibility, generally between 1 and 50 amino acid residues in length, but not limited to this. In some cases, as long as it does not affect the two components being connected to perform their intended functions, any amino acid residue chain between the two components can be regarded as a peptide linker. Therefore, in some aspects, the heterodimeric Fc scaffold according to the present invention, or the CH3 heterodimer according to the present invention, is connected to a polypeptide of interest, such as a target binding domain according to the present invention, or other functional polypeptide or protein domain, via a peptide linker, all under consideration in the present invention.

[0134] In the heterologous multimeric protein according to the present invention, those skilled in the art can easily determine the available linker sequence based on the components to be connected and the connection position. In some embodiments, the linker is used to connect the Fc scaffold and the antigen binding domain according to the present invention; and / or to connect two antigen binding domains. In some embodiments, the available linker is a flexible connecting peptide of 5-50 amino acids, preferably comprising glycine (G) and / or serine (S) and / or threonine residues (T). In some embodiments, the linker has a length of 5-30 amino acids, for example, 8, 10, 15, 20, 25 or 30 amino acids in length, or has an amino acid length falling between any two integers. In some embodiments, the linker comprises the amino acid sequence (G4S) n, wherein n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7. In a preferred embodiment, the linker consists of the amino acid sequence (G4S)2. In other embodiments, the linker is a hinge region from an immunoglobulin or a derivative thereof. Examples of other useful linkers include, for example, but are not limited to, the following amino acid sequences: (Gly3Ser)2 (SEQ ID NO: 22), (Gly4Ser)2 (SEQ ID NO: 12), (Gly3Ser)3 (SEQ ID NO: 24), (Gly4Ser)3 (SEQ ID NO: 25), (Gly3Ser)4 (SEQ ID NO: 26), (Gly4Ser)4 (SEQ ID NO: 27), (Gly3Ser)5 (SEQ ID NO: 28), (Gly4Ser)5 (SEQ ID NO: 29), (Gly3Ser)6 (SEQ ID NO: 30), (Gly4Ser)6 (SEQ ID NO: 31), GGG (SEQ ID NO: 32), DGGGS (SEQ ID NO: 33), TGEKP (SEQ ID NO: 34), GGRR (SEQ ID NO: 35), EGKSSGSGSESKVD (SEQ ID NO: 36). NO: 36), KESGSVSSEQLAQFRSLD (SEQ ID NO: 37), GGRRGGGS (SEQ ID NO: 38), LRQRDGERP (SEQ ID NO: 39), LRQKDGGGSERP (SEQ ID NO: 40) and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23). Alternatively, if necessary, a computer program can be used to simulate the three-dimensional structure of proteins and peptides, or a phage display method can be used to rationally design a suitable flexible linker peptide.

[0135] In a preferred embodiment, a peptide linker having the sequence shown in SEQ ID NO: 12 is used to connect the Fc scaffold and its N-terminal antigen binding domain, such as VHH or Fab.

[0136] In some cases, chemically synthesized linkers can also be used to connect the binding domain to the Fc scaffold. In this case, the connection is also called "conjugation". Examples of chemically synthesized linkers include: N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl suberate) (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (Sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (Sulfo-DST), bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfosuccinimidyloxycarbonyloxy)ethyl]sulfone (Sulfo-BSOCOES), etc.

[0137] When multiple linkers are used in the heteromultimeric proteins of the present invention, it is understood that these linkers can be the same as or different from each other.

[0138] In some cases, chemically synthesized linkers can also be used to conjugate chemically synthesized therapeutic agents (e.g., toxin small molecules) or diagnostic agents to heterologous multimeric proteins of the present invention, such as bispecific antibodies, such as sugar chains of the CH2 domain. Such conjugates are also contemplated by the present invention.

[0139] Exemplary heteromultimeric proteins

[0140] In some embodiments, the heteromultimeric proteins of the present invention are multispecific binding proteins, particularly bispecific antibodies, comprising binding specificities for different antigenic epitopes. Bispecific antibodies with binding specificities for at least two different antigens or antigenic epitopes have broad clinical potential and can be used as targeted drugs for in vitro and in vivo immunodiagnosis and therapy, as well as in diagnostic immunoassays. See, for example, WO9850431A2. Bispecific antibodies can be used in in vitro assays to probe the functional properties of cell surface molecules and determine cytotoxicity mediated by different Fc receptors (Fanger et al., Crit. Rev. Immunol. 12: 101-124 (1992)); for enzyme-linked immunosorbent assays (Nolan et al., Biochem. Biophys. Acta. 1040: 1-11 (1990); Hammerling et al, J. Exp. Med. 128: 1461-1473 (1968)); and in vitro or in vivo immunodiagnosis of various diseases such as cancer (Songsivilai et al., Clin. Exp. Immunol. 79: 315 (1990)). To facilitate the diagnostic use of BsAbs, one arm of the BsAb can bind to a diseased tissue or cell surface antigen (e.g., a tumor-associated antigen), while the other arm can bind to a detectable label, such as a chelator that tightly binds to a radionuclide. In therapeutic applications, bispecific antibodies can be used to direct the patient's cellular immune defense mechanism to specifically target diseased tissues / cells, such as tumor cells, or pathogens. To this end, one arm of the BsAb can bind to an antigen on the surface of an immune cell (e.g., T cell, NK cell), while the other arm can bind to an antigen on the surface of the diseased tissue / cell.

[0141] In some embodiments, the multispecific antibody according to the present invention is a heterodimer consisting of or essentially consisting of a heterodimeric Fc scaffold according to the present invention and at least one antigen binding domain attached thereto, having a configuration selected from the following:

[0142] -(antigen-binding domain)n-Fc / (antigen-binding domain)n-Fc,

[0143] -(antigen-binding domain)n-Fc-(antigen-binding domain)m / (antigen-binding domain)n-Fc,

[0144] -(antigen-binding domain)n-Fc-(antigen-binding domain)m / (antigen-binding domain)n-Fc-(antigen-binding domain)m,

[0145] wherein each of n and m is independently selected from integers of 0, 1 and 2;

[0146] Each of the above antigen binding domains is independently selected from the group consisting of: Fv, scFv, Fab, scFab, crossFab, VHH, ligand binding domain of a ligand or receptor;

[0147] The symbol “-Fc” indicates attachment at the N-terminus of Fc;

[0148] The notation "Fc-" indicates attachment at the C-terminus of Fc;

[0149] Here, the two monomers of the heterodimer are separated by the symbol “ / ”;

[0150] Among them, one of the two Fc chains is a knob chain, and the other is a hole chain.

[0151] In some preferred embodiments, n is 1 and m is 0. In some preferred embodiments, n is 0 and m is 1. In some preferred embodiments, n is 1 and m is 1. In some preferred embodiments, each of the above antigen binding domains is independently selected from the group consisting of: scFv, VHH, and Fab.

[0152] In some specific embodiments, the multispecific heterodimeric proteins according to the present invention are bispecific and have an IgG or IgG-like configuration.

[0153] In some specific embodiments, the multispecific heterodimeric protein according to the present invention is a bispecific antibody and has a configuration selected from the following:

[0154] -scFv-Fc / scFv-Fc, wherein scFv domains for two different specificities are respectively linked to the N-terminus of the Fc scaffold according to the invention;

[0155] -Fab-Fv / Fab-Fc, wherein the Fab domains for two different specificities are respectively linked to the N-terminus of the Fc scaffold according to the invention;

[0156] - VHH-Fc / VHH-Fv, wherein the VHH domains for two different specificities are respectively linked to the N-terminus of the Fc scaffold according to the invention;

[0157] - scFv-Fc / Fab-Fc, wherein the scFv domain and the Fab domain for two different specificities are respectively linked to the N-terminus of the Fc scaffold according to the invention;

[0158] -scFv / VHH-Fc, wherein the scFv domain and the VHH domain for two different specificities are respectively connected to the N-terminus of the Fc scaffold according to the present invention;

[0159] - VHH / Fab-Fc, wherein the VHH domain and the Fab domain for two different specificities are respectively linked to the N-terminus of the Fc scaffold according to the invention.

[0160] In embodiments where the heteromultimeric protein according to the present invention comprises two or more Fab domains with different specificities, for example, when the heteromultimeric protein is a bispecific antibody with a Fab-Fc / Fab-Fc configuration, light chain mispairing can be prevented by using a common light chain. See, for example, US Pat. No. 7,951,917B. Alternatively, a first Fab-Fc half antibody and a second Fab-Fc half antibody can be produced separately, followed by in vitro assembly of the desired heterodimer by adding an appropriate reducing agent to the mixture.

[0161] In some embodiments, the heterologous multimeric protein of the present invention is a bispecific antibody comprising a VHH domain and a Fab domain that bind to different antigens, respectively. In some embodiments, the bispecific antibody binds to HSA and B7-H3. In some embodiments, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of the VHH amino acid sequence set forth in SEQ ID NO: 11; or comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 95% identity thereto. In some embodiments, the Fab domain comprises a VH and a VL, wherein the VH comprises the CDR1, CDR2, and CDR3 sequences of the VH amino acid sequence set forth in SEQ ID NO: 13; or comprises the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having at least 95% identity thereto; and wherein the VL comprises the CDR1, CDR2, and CDR3 sequences of the VL amino acid sequence set forth in SEQ ID NO: 14; or comprises the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 95% identity thereto. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 11; and the Fab comprises the VH of SEQ ID NO: 13 and the VL of SEQ ID NO: 14. In some embodiments, the bispecific antibody is a bivalent antibody. In some embodiments, the VHH and Fab domains are respectively linked to the N-termini of the first and second polypeptides of the heterodimeric Fc scaffold according to the present invention.

[0162] In some embodiments, the binding protein of the present invention is a bispecific antibody comprising a first and a second Fab domain that bind to different antigens, respectively. In some embodiments, the bispecific antibody binds to EGFR / HER2. In some embodiments, the first Fab domain comprises a VH and a VL, wherein the VH comprises the CDR1, CDR2, and CDR3 sequences of the VH amino acid sequence set forth in SEQ ID NO: 15; or comprises the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least 95% identity thereto; and wherein the VL comprises the CDR1, CDR2, and CDR3 sequences of the VL amino acid sequence set forth in SEQ ID NO: 16; or comprises the amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence having at least 95% identity thereto. In some embodiments, the second Fab domain comprises a VH and a VL, wherein the VH comprises the CDR1, CDR2, and CDR3 sequences of the VH amino acid sequence set forth in SEQ ID NO: 17; or comprises the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence having at least 95% identity thereto; and wherein the VL comprises the CDR1, CDR2, and CDR3 sequences of the VL amino acid sequence set forth in SEQ ID NO: 18; or comprises the amino acid sequence set forth in SEQ ID NO: 18, or an amino acid sequence having at least 95% identity thereto. In some embodiments, the first Fab domain comprises the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16; and the second Fab domain comprises the VH of SEQ ID NO: 17 and the VL of SEQ ID NO: 18. In some embodiments, the bispecific antibody is a bivalent antibody. In some embodiments, the first and second Fab domains are attached to the N-termini of the first and second polypeptides, respectively, of the heterodimeric Fc scaffold according to the present invention.

[0163] IV. Polynucleotides, Vectors, and Host Cells

[0164] In some aspects, the present invention provides one or more polynucleotides encoding a CH3 heterodimer, a heterodimer Fc scaffold, a heteromultimeric protein or a binding protein according to the present invention; one or more vectors comprising the one or more polynucleotides, wherein the one or more polynucleotides may be present in a single vector or in separate multiple vectors. In other aspects, the present invention also provides a host cell comprising the one or more nucleic acids or the one or more vectors. Depending on the circumstances, a single or multiple vectors comprising the one or more nucleotides may be introduced into the same host cell to express and produce a target product in one host cell; or a single or multiple vectors comprising the one or more polynucleotides may be introduced separately into different host cells to express and produce intermediates comprising different chains or different chain combinations (e.g., different monomeric proteins) of a heteromultimer according to the present invention in different host cells, and under conditions suitable for assembling a heteromultimer according to the present invention, by mixing the intermediates, a heteromultimer according to the present invention is produced.

[0165] In the present invention, the expression vectors that can be used are not particularly limited, and include but are not limited to viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0166] In the present invention, the host cell that can be used is not particularly limited. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells, such as mammalian host cells, insect cells, etc. The example of useful mammalian host cell line includes monkey kidney CV1 system (COS-7) transformed by SV40, human embryonic kidney system (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2 / 0, etc. In some embodiments, a mammalian cell line suitable for suspension culture can be used. In some preferred embodiments, the host cell is CHO or HEK293 cells.

[0167] V. Production and Purification of Heteromultimeric Proteins of the Invention

[0168] In another aspect, the present invention provides a method for producing a heteromultimeric protein of the present invention. To produce a heteromultimeric protein of the present invention, the polypeptide chains of the heteromultimeric protein of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production, and assembled under appropriate conditions. As previously described, in the case of recombinant production, the assembly can occur in a host cell used to express the heteromultimeric protein; or, if necessary, the assembly of the heteromultimeric protein can be performed in vitro after harvesting the expressed intermediate polypeptide chains from the host cell.

[0169] In one embodiment, therefore, the present invention provides a method for producing a heteromultimeric protein of the present invention, the method comprising: culturing a host cell comprising a protein encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the heteromultimeric protein; and assembling the polypeptide chains to produce the heteromultimeric protein under conditions suitable for the assembly of the polypeptide chains into the heteromultimeric protein.

[0170] In some embodiments, the method comprises: culturing a host cell encoding a polypeptide chain of the heteromultimeric protein under conditions suitable for expressing the polypeptide chain of the heteromultimeric protein; and recovering the heteromultimeric protein produced by the host cell from the cell culture. Preferably, the recovered product, after Protein A affinity chromatography, is assayed by SEC-HPLC to determine that the purity of the heteromultimeric protein according to the present invention is greater than 80%, preferably greater than 85%, and more preferably greater than 90%.

[0171] In other embodiments, the method comprises: culturing a host cell encoding a polypeptide chain of the heteromultimeric protein under conditions suitable for expressing the polypeptide chain of the heteromultimeric protein; and recovering intermediates of the heteromultimeric protein produced by the host cell from the cell culture; mixing the intermediates under conditions suitable for assembly to produce the heteromultimeric protein, and recovering the resulting heteromultimeric protein. In the case of a heterodimeric protein, monomers comprising the knob chain and monomers comprising the hole chain are expressed separately in different host cells, and the heteromultimeric protein is produced and recovered from the monomer mixture under conditions suitable for assembly. Preferably, the recovered product, after ion exchange chromatography and as determined by SEC-HPLC, has a purity of greater than 80%, preferably greater than 85%, and more preferably greater than 90%.

[0172] Antibodies produced by the methods described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. After purification, the purity of the heteromultimeric proteins of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the heteromultimeric proteins provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0173] VI. Compositions and Uses

[0174] The present invention also relates to a composition comprising a heteromultimeric protein of the present invention (e.g., a binding protein according to the present invention). In embodiments where the composition is a pharmaceutical composition, the composition further comprises a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delaying agents.

[0175] In the present invention, a pharmaceutical composition generally refers to a drug used for the treatment or prevention, or for the detection or diagnosis of a disease. In some preferred embodiments involving the pharmaceutical compositions of the present invention, the heteromultimeric protein of the present invention is a bispecific antibody, for example, a bispecific antibody in which at least one of the antibodies specifically targets a disease-associated antigen. In some embodiments, the heteromultimeric protein of the present invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise the heteromultimeric protein described herein and one or more other therapeutic agents.

[0176] The pharmaceutical composition of the present invention can be prepared into a formulation according to methods well known to those skilled in the art. In addition, it is contemplated to use pharmaceutically acceptable carriers or media, such as sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, solvents, preservatives, binders, etc., in appropriate combination with the heterologous multimeric protein of the present invention to formulate a unit dosage form that meets the needs of drug administration. For example, a pharmaceutically acceptable carrier can be used to prepare a sterile solution or suspension for parenteral administration in the form of an injection. The amount of active ingredient in the formulation will be set to produce an effective amount within a predetermined range after administration to an individual.

[0177] Pharmaceutical composition of the present invention is suitable for various routes of administration, including but not limited to intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (for example, by injection or infusion). Accordingly, pharmaceutical composition of the present invention can be formulated into the dosage form applied to expected route of administration, for example, injection dosage form, nasal administration dosage form, through lung administration dosage form, transdermal administration dosage form. For injection dosage form, route of administration can include, but is not limited to, systemic or local administration such as intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intratumoral injection. In addition, the specific administration method of pharmaceutical composition of the present invention can be suitably selected according to the age, symptom of patient.

[0178] In addition, detection or diagnostic kits comprising the heteromultimeric proteins described herein are also within the scope of the present invention. The kits may include one or more other elements, such as instructions for use; other reagents, such as labels or reagents for conjugation; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0179] The following examples are described to assist understanding of the present invention. The examples are not intended to, and should not be interpreted in any way as, limiting the scope of protection of the present invention. Example

[0180] Example 1 Design of Fc mutation candidate combination sequences

[0181] The researchers searched the Protein Data Bank (www.pdb.org) for knob-into-hole (KIH) Fc heterodimer crystal structures (e.g., 4NQS and 5ID8) and compared them with wild-type IgG1 Fc crystal structures (e.g., 1HZH) to analyze the reasons for the reduced thermal stability of the KIH model. They found that the KIH mutation reduced the number and strength of the amino acid contacts between the two CH3 domains.

[0182] In wild-type IgG1 Fc, key amino acids at the CH3-CH3 domain interface, including L351, L368, and Y407, form a hydrophobic core, covering a large surface area (see Figure 1A). However, in the KIH model, the surface area of ​​the hydrophobic core is significantly reduced due to the mutations of L368A and Y407V to amino acids with small side chains (see Figure 1B). Furthermore, the interactions between some amino acids near the hydrophobic core are also affected. For example, in wild-type IgG1 Fc, the side chain of K409 in one CH3 domain directly contacts amino acids L368, K370, D399, F405, and Y407 in another CH3 domain. In particular, K409 forms a complex hydrogen bond network with D399, S364, T411, and a water molecule, while preventing electrostatic clashes between K409 and K370. However, in the KIH structure, the K409 side chain in the Knob chain is affected by the T366W mutation, losing direct contact with L368A, Y407V, and also losing the stabilizing water molecules and the hydrogen bond network mediated by them.

[0183] To enhance CH3 interaction strength and improve thermal stability, we selected amino acids near the Fc contact interface in the KIH model as targets for mutation. We ultimately designed a series of Fc heterodimer mutation combinations. The specific amino acid mutation information is shown in Table 1. The KIH combination is a handle-hole mutation combination published in US Patent No. 7951917B1. M1-M7 are newly designed candidate mutation combinations.

[0184] Table 1. Design of Fc heterodimer mutation combinations, where chain A is the chain containing the knob mutation and chain B is the chain containing the corresponding hole mutation

[0185] Example 2 Expression and purification of VHH-Fc / Fc heterodimers

[0186] In this example, to verify the effect of the Fc mutant combinations listed in Table 1 on promoting heterodimer formation, A-chain VHH-Fc fusion molecules and B-chain Fc molecules were first designed. Furthermore, VHH-Fc and Fc genes were synthesized, and expression plasmids were constructed. After transfection into 293F cells, the culture supernatant was collected and purified by Protein A affinity. The purified product was then analyzed for heterodimer and other impurities using SDS-PAGE and SEC-HPLC to determine the effect of different mutation combinations on the expression yield and purity of the VHH-Fc / Fc heterodimer.

[0187] Expression vector construction

[0188] Based on the amino acid sequence of the human IgG1 constant region (P01857) from the Uniprot protein database, the human IgG1-Fc amino acid sequence, comprising the hinge region, CH2 domain, and CH3 domain, was derived. Ten Fc mutant amino acid sequences were then designed according to Table 1 (see SEQ ID NOs: 1 to 10). A VHH fragment (derived from the ozoralizumab antibody, see SEQ ID NO: 11) and a flexible linker polypeptide (see SEQ ID NO: 12) were introduced at the N-terminus of the A-chain Fc fragment to construct the A-chain VHH-Fc fusion molecule; the B-chain Fc fragment remained unchanged. The molecular weight difference between the VHH-Fc and Fc facilitates differentiation of the proportions of AB heterodimers, AA / BB homodimers, A / B monomers, or high-molecular-weight aggregates (HMWSs) formed during recombinant expression.

[0189] The A-chain VHH-Fc and B-chain Fc DNA encoding genes were designed using codons optimized for mammalian / human expression and cloned into the expression vector pcDNA3.4 (Invitrogen) to generate plasmids for expressing VHH-Fc and Fc. Gene synthesis and plasmid construction were performed by Genwiz (Suzhou).

[0190] 293F cell transfection

[0191] VHH-Fc and Fc plasmids were mixed at a molar ratio of 1:1 or 0.6:1, and then added to 1 / 10 the transfection volume of OPM-CD Trans293 medium (OPM, P82019). FectoPRO transfection reagent (PolyPlus, PT-116-010) was added to the medium at a ratio of 1 μl per 1.2 ml of cells. The medium containing the plasmids and transfection reagent was mixed and allowed to stand for 10 minutes. The mixture was then added to a shake flask containing Expi293F cells (Invitrogen, A14635) and cultured with shaking at 37°C and 8% CO2. Sixteen hours after transfection, feed supplement (OPM, F081918-001) at 10% of the transfection volume was added, and culture was continued for 6 days.

[0192] Protein A purification

[0193] A 1 ml Protein A Diamond chromatography column (Borgron, AA0273) was equilibrated with 5 column volumes of equilibration solution (20 mM sodium phosphate, pH 7.4). The clarified cell culture medium was loaded onto the column at a flow rate of 0.5 column volumes / min. The column was then rinsed sequentially with 5 column volumes of equilibration solution (20 mM sodium phosphate, pH 7.4) and pre-elution solution (20 mM acetic acid-sodium acetate, pH 5.2). Finally, elution was performed with 10 column volumes of eluent (20 mM acetic acid-sodium acetate, pH 3.2). The collected eluate was adjusted to pH 6.0 with 1.5 M Tris base.

[0194] SDS-PAGE analysis

[0195] 5 μg of Protein A column purified product was mixed with non-reducing loading buffer (without DTT) and run on a 10% SurePAGE gel (Genscript, M00666). After electrophoresis, the gel was stained with Coomassie Brilliant Blue and destained, and photographed using a gel imager.

[0196] The results are shown in Figure 2. The co-expression product of the VHH-Fc plasmid and the Fc plasmid primarily contained five proteins of varying molecular weights: AB heterodimer, AA homodimer, BB homodimer, A monomer, and B monomer. At a plasmid ratio of 1:1, the heterodimer content of the KIH control, M1, M2, M5, and M6 combinations was high, exceeding 90%, while the heterodimer content of the M3, M4, and M7 combinations was lower, ranging from 80% to 90%. At a plasmid ratio of 0.6:1, the heterodimer content of the M1, M3, M5, M6, and M7 combinations was high, exceeding 90%, while the heterodimer content of the KIH, M2, and M4 combinations was lower, ranging from 80% to 90%. These results suggest that, compared with KIH, the M1 and M6 combinations have a greater ability to promote AB heterodimer formation and inhibit AA / BB homodimer formation. The M2 combination had similar abilities to promote AB heterodimer and AA / BB homodimer formation as the KIH control. The M3 combination had similar abilities to promote AB heterodimer formation as the KIH combination, but induced more AA homodimers and less BB homodimers.

[0197] SEC-HPLC analysis

[0198] The 1 mg / ml Protein A column-purified product was subjected to SEC analysis using a Theromo Vanquish Core HPLC instrument. The chromatographic column was a Waters BioResolve SEC mAb (2.5 μm, 7.8 × 300 mm). Isocratic elution was performed with PBS as the mobile phase. The elution time was 30 minutes, the flow rate was 0.5 ml / min, the detection wavelength was 280 nm, the column temperature was 20°C, the injection volume was 10 μl, and the temperature of the thermostat was 5°C.

[0199] The results are shown in Table 2. Overall, the dimer ratios calculated by SEC-HPLC analysis for each mutant combination were similar to those found by SDS-PAGE analysis. At a plasmid ratio of 1:1, the heterodimer content of the KIH control, M1, M2, M5, and M6 combinations was high, exceeding 90%, but the heterodimer content of the M3, M4, and M7 combinations was low, ranging from 80% to 90%. At a plasmid ratio of 0.6:1, the heterodimer content of the M1, M3, M5, M6, and M7 combinations was high, exceeding 90%, but the heterodimer content of the KIH, M2, and M4 combinations was low, ranging from 80% to 90%.

[0200] Table 2 SEC-HPLC analysis of VHH-Fc / Fc mutant combinations

[0201] Combined with the above results, it can be seen that introducing the hole L351Y mutation based on KIH does not substantially change the ratio of heterodimers to homodimers. Introducing the K409D / D399R double mutation based on KIH-L351Y does not substantially change the heterodimer ratio, but induces more AA homodimers and fewer BB homodimers.

[0202] Example 3 Thermal Stability Analysis of VHH-Fc / Fc Heterodimers

[0203] Heterodimer purification

[0204] A 1ml Mono S ion exchange column (Cytiva, 17516801) was flushed with equilibration solution (20mM sodium phosphate, pH 6.0) for 5 column volumes or to baseline. Protein A affinity-purified samples were adjusted to pH 6.0 with 1.5M Tris base or 50mM sodium acetate (pH 3.2) and loaded onto the Mono S column at a flow rate of 0.5 column volume / min. The column was then flushed with equilibration solution for 10 column volumes. Finally, the column was eluted with eluent (20mM sodium phosphate + 1M sodium chloride, pH 6.0) at a gradient of 0-30%, and the eluate was collected in separate tubes. The heterodimer purity of each tube was determined by SEC-HPLC, and samples with a purity greater than 98% were combined.

[0205] Thermal stability test

[0206] The thermal stability of the samples was analyzed using a microcal differential scanning calorimeter (Microcal PEAQ-DSC). The method was as follows: the protein sample was diluted to 1 mg / mL in PBS buffer. The assay temperature range was set between 20°C and 100°C, with a heating rate of 90°C / h. PBS was added to the assay cell as a blank buffer, and a temperature scan was performed. The solution in the sample cell was then removed, and the sample to be tested was added, and a temperature scan was performed. The experimental data was processed using the instrument's built-in analysis software to calculate the Tm value.

[0207] The Tm measurements of the VHH-Fc / Fc mutant combinations are shown in Table 3 and Figure 3. The KIH control exhibited at least two Tm values: the first (Tm1) of 65.68°C, reflecting the thermal stability of the VHH fragment and CH2 domain, and the second (Tm2) of 71.48°C, reflecting the thermal stability of the CH3 domain. The Tm values ​​of the M1 and M6 mutant combinations were only 67.56°C and 66.31°C, respectively, significantly lower than those of the KIH control. These results suggest that the introduction of electrostatic steering into the KIH structure unexpectedly reduces the thermal stability of the CH3 domain. Interestingly, the CH3 domain Tm of the M2 mutant combination reached approximately 74.03°C, an increase of approximately 2.5°C compared to the KIH control; the CH3 domain Tm of the M3 mutant combination was even higher, reaching approximately 75.84°C. The results suggest that a single amino acid mutation, L351Y, in Fc chain B can improve CH3 domain stability in the KIH format. Furthermore, this mutation, combined with the amino acid mutations K409D in Fc chain A and D399R in Fc chain B, exhibits a synergistic effect, further enhancing CH3 domain stability. Other mutant combinations, including M4, M5, and M7, exhibited poor thermal stability, with Tm values ​​below 70°C. These results suggest that introducing either L351Y in chain A or L351Y-D399K in chain B into the KIH construct does not improve CH3 domain thermal stability.

[0208] Table 3 Tm values ​​of VHH-Fc / Fc mutant combinations

[0209] Example 4 Expression and purification of VHH-Fc / Fab-Fc heterodimers

[0210] To verify the versatility of the mutation combination in Example 2 across different antibody sequences and molecular formats, a VHH-Fc / Fab-Fc heterodimer was constructed. A VHH antibody fragment (amino acid sequence shown in SEQ ID NO: 11) and a flexible linker polypeptide (see SEQ ID NO: 12) were introduced into the N-terminus of the first Fc fragment to form an A-chain VHH-Fc fusion protein. A Fab antibody fragment was introduced into the N-terminus of the second Fc fragment to form a Fab-Fc half-antibody. The Fab amino acid sequence was derived from the B7-H3 mouse monoclonal antibody obtained through hybridoma technology and contains the light chain domain VL-CL (amino acid sequence shown in SEQ ID NO: 14) and the heavy chain domain VH-CH1 (amino acid sequence shown in SEQ ID NO: 13). The heavy chain of the Fab-Fc antibody is designated as the B chain, and the light chain is designated as the C chain. The molecular weight difference between VHH-Fc fusion proteins and Fab-Fc half antibodies facilitates the differentiation of various protein components formed during recombinant expression, including ABC heterodimers, AA homodimers, BB homodimers, BBCC homodimers, A monomers, B monomers, and C monomers. The target bispecific antibody is the ABC heterodimer, while the others are impurities.

[0211] Similar to that described in Example 2, the A chain, B chain, and C chain genes were synthesized, plasmids expressing the A chain, B chain, and C chain, respectively, were constructed, and 293F cells were transfected. The culture supernatant was collected, Protein A affinity purified, and the purified products were analyzed for the content of ABC heterodimers and other impurities using SDS-PAGE and SEC-HPLC methods.

[0212] SDS-PAGE analysis results are shown in Figure 4. Two different plasmid transfection ratios (A-chain plasmid:B-chain plasmid:C-chain plasmid) were used: 1:1:1.5 or 0.6:1:1.5. The KIH control exhibited less than 90% ABC heterodimer purity under both conditions. The other mutant combinations, M1, M2, M3, M5, M6, and M7, exhibited ABC heterodimer purity exceeding 90% under at least one condition. However, the M4 mutant combination was less effective, with ABC heterodimer purity lower than that of the KIH control under both conditions.

[0213] The results of SEC-HPLC analysis are shown in Table 4. Under both ABC plasmid ratios of 1:1:1.5 and 0.6:1:1.5, the expression levels of the M1-M7 mutant combinations were greater than 200 mg / L, similar to or higher than the KIH control. The ABC heterodimer ratios calculated by SEC-HPLC for each mutant combination were generally similar to those found in SDS-PAGE analysis. The ABC heterodimer purity of the KIH control was less than 90% under both conditions. Under an ABC plasmid ratio of 1:1:1.5, the ABC heterodimer ratios of the M1-M7 mutant combinations were higher than those of the KIH control, with the heterodimer ratios of M1, M2, M3, M5, and M7 exceeding 90%. Under an ABC plasmid ratio of 0.6:1:1.5, the heterodimer ratios of the M1, M6, and M7 combinations were higher than those of the KIH control and greater than 90%.

[0214] Table 4 SEC-HPLC analysis of VHH-Fc / Fab-Fc mutant combinations

[0215] Example 5 Expression and purification of Fab-Fc half antibody

[0216] To verify the versatility of the mutation combination of Example 2 across different antibody sequences and molecular formats, two Fab fragments binding to different antigens were also linked to the Fc region to construct two different Fab-Fc half-antibody mutants. The A chain Fc designed in Table 1 of Example 1 was linked to the first Fab at the N-terminus to obtain the A half-antibody, wherein the Fab was derived from the anti-EGFR monoclonal antibody Zalutumumab and contained the heavy chain domain VH-CH1 (amino acid sequence shown in SEQ ID NO: 15) and the light chain domain VL-CL (amino acid sequence shown in SEQ ID NO: 16). The B chain Fc designed in Table 1 of Example 1 was linked to the second Fab at the N-terminus to obtain the B half-antibody, wherein the Fab fragment was derived from the anti-HER2 monoclonal antibody Pertuzumab and contained the heavy chain domain VH-CH1 (amino acid sequence shown in SEQ ID NO: 17) and the light chain domain VL-CL (amino acid sequence shown in SEQ ID NO: 18).

[0217] Similar to that described in Example 2, the light chain and heavy chain genes of half antibodies A and B were synthesized, plasmids expressing the genes were constructed, and 293F cells were transfected to express half antibody A and half antibody B, respectively. The culture supernatant was collected, affinity purified by Protein A, and the purified products were analyzed by SDS-PAGE and SEC-HPLC methods.

[0218] SDS-PAGE analysis results are shown in Figure 5. For all half-antibody mutants tested, the 293 cell expression products primarily formed half-antibody monomers with a molecular weight of 75 kDa and homodimers with a molecular weight of 150 kDa, along with small amounts of light chains with a molecular weight of 25 kDa and heavy chains with a molecular weight of 50 kDa. The monomer ratio for the Fab-Fc mutants K0, K2, K7, H0, H2, and H7 was greater than 80%, while the monomer ratio for mutants K9 and H8 was less than 50%.

[0219] The results of SEC-HPLC analysis are shown in Table 5. All half-antibody mutants expressed at levels greater than 100 mg / L in 293 cells. The proportions of half-antibody monomer, homodimer, and multimer in the 293 cell expression products were calculated using the SEC-HPLC analysis software. The combined monomer and dimer content of all Fab-Fc mutants was greater than 95%, and the multimer content was less than 5%. K0 produced 46.27% dimer, and K2 produced 56.17% dimer, which likely included both non-covalent dimers and disulfide-linked covalent dimers. This result suggests that the K409D mutation has little effect on CH3 domain homodimerization. K9 produced approximately 86.2% dimer. K9 produced significantly more dimers than K0, suggesting that the L351Y mutation promotes CH3 domain homodimerization. K7 produced 26.51% dimer and 73.24% monomer. H0 produced 30.4% dimers, which should include both non-covalent and disulfide-linked covalent dimers. H2 produced 14.38% dimers, and H7 produced 15.05%. H2 and H7 produced significantly fewer homodimers than H0, suggesting that the D399R mutation prevents CH3 domain homodimerization through electrostatic repulsion. H8 produced 64.71% dimers. H8 produced significantly more dimers than H0, suggesting that the L351Y mutation promotes CH3 domain homodimerization.

[0220] Table 5 SEC-HPLC analysis results of Fab-Fc half antibody

[0221] Example 6 Preparation and Purification of Fab-Fc / Fab-Fc Heterodimers

[0222] In vitro assembly

[0223] The half-antibody solution obtained by affinity chromatography in Example 5 was mixed with the corresponding half-antibody combination shown in Table 6 at an equimolar ratio (1:1). The mixture was then neutralized to pH 8-8.2 with 1.5 M Tris base. The total protein content of the reaction system was 2 mg. After standing at room temperature for 30 minutes, a 200x molar ratio GSH solution (Sigma, S0073) was added. The reaction sample was incubated at 37°C for 4 hours. Samples were taken at 0.5, 1, 2, and 4 hours and immediately assayed for in vitro assembly efficiency. The reaction sample was transferred to a 4°C refrigerator and allowed to stand overnight.

[0224] HIC-HPLC (hydrophobic interaction high performance liquid chromatography) analytical purity

[0225] HIC testing was performed using a Theromo Vanquish Core high-performance liquid chromatograph. The chromatographic column was TOSOH TSKgel Bulty-NPR (specification: 2.5 μm, 4.6 × 100 mm). A mixed solution of 1.5 M ammonium sulfate and 20 mM phosphate (pH 7.0) was used as mobile phase A, and a 20 mM phosphate solution (pH 7.0) was used as mobile phase B for gradient elution (0-15 min 0% B-100% B, 15.5-22 min 0% B-0% B). The flow rate was 0.6 ml / min, the detection wavelength was 280 nm, the column temperature was 25°C, the injection volume was 10 μl, and the temperature of the thermostat was 5°C.

[0226] Figure 6 shows the HIC-HPLC chromatograms of the half-antibody and the bispecific antibody after a 4-hour reaction. This method allows for sensitive detection of the assembly efficiency of bispecific heterodimers. Differences in hydrophobicity between the monomers or homodimers formed by the half-antibody result in different peak times in the HIC chromatogram. The bispecific antibody molecules generated by in vitro assembly of two Fab-Fc molecules also exhibit different peak times than the half-antibody.

[0227] The results of HIC-HPLC analysis are shown in Table 6. After incubation at 37°C for 0.5 hours, the proportion of AB heterodimers produced by the combination of KIH, M1, M2, and M3 mutants was already greater than 90%. After incubation at 37°C for 2 and 4 hours, the proportion of AB heterodimers produced by the combination of KIH, M1, M2, and M3 mutants increased to approximately 95%.

[0228] Table 6 HIC-HPLC analysis of bispecific antibody assembly efficiency

[0229] Heterodimer purification

[0230] A 1ml Capto S ImpAct ion exchange column (Cytiva, 17371751) was rinsed with equilibration solution (20mM sodium phosphate, pH 6.0) for 5 column volumes or to baseline. The in vitro assembled sample was adjusted to a pH of 5.8-6.2 with 1M acetic acid, then diluted 1-2-fold with ultrapure water and filtered through a 0.22µm PES filter. The sample was loaded onto the Capto S column at a flow rate of 0.5 column volumes / min. The column was then rinsed with equilibration solution for 10 CV. Finally, the column was eluted with eluent (20mM sodium phosphate + 1M sodium chloride, pH 6.0) using a 0-30% gradient, and the eluate was collected in separate tubes. The purity of the dimer peak in each tube was determined by SEC-HPLC, and samples with a purity greater than 98% were combined.

[0231] The results of SEC-HPLC analysis are shown in Table 7. After incubation at 37°C for 4 hours, the dimer ratio of the combined KIH, M1, M2, and M3 mutants was greater than 96%, with the AB heterodimer being the major component and AA or BB homodimers being minor. Other impurities included A or B monomers or high-molecular-weight aggregates, all accounting for less than 2%.

[0232] Table 7 SEC-HPLC analysis of the purity of the bispecific antibodies produced by incubation at 37°C for 4 h

[0233] Example 7 Bisspecific Antibody Binding Activity to Antigen

[0234] The binding activity of the purified bispecific antibodies in Example 6 to the antigen was examined. Recombinant human EGFR protein (ACRO Biosystems, EGR-H5222) and HER2 protein (ACRO Biosystems, HE2-H5225) were diluted to 1 μg / ml with coating buffer (Solarbio, C1055). 100 μl was added to each well of the plate and incubated at 4°C for 16 hours. The blocking agent was prepared by adding 3% BSA (Sangong, 9048-46-8) and 0.05% Tween 20 (Sangong, A600560-0500) to a PBS solution. After washing the plate three times with 0.05% PBST, 300 μl of blocking agent was added to each well and allowed to stand at room temperature for 2 hours. The bispecific antibodies were diluted with 0.5% BSA-0.05% PBST blocking buffer to a maximum concentration of 15 μg / ml, using a 3-fold continuous gradient. After washing the plate three times with 0.05% PBST, 100 μl of double antibody solution was added to each well and incubated at room temperature for 1 hour. After washing the plate three times with 0.05% PBST, 100 μl of HRP-conjugated secondary antibody (Bethyl, A80-304P, 1:5000 dilution) was added to each well and incubated at room temperature for 1 hour. After washing the plate three times with 0.05% PBST, 100 μl of color development solution (Solarbio, PR1210) was added to each well and incubated at room temperature in the dark for 10 minutes. Then, 100 μl of stop solution (Solarbio, C1058) was added to each well and absorbance at OD450 was read using a multi-function microplate reader (Tecan, Spark).

[0235] ELISA results are shown in Figure 7. The bispecific antibodies generated by combining the KIH, M1, M2, and M3 mutants exhibited very similar antigen-binding curves. IC50 values ​​for EGFR binding ranged from 17.0 to 24.9 ng / ml, and for HER2 from 12.8 to 14.6 ng / ml. These results demonstrate that mutations in the CH3 domain of the Fc region do not affect the antigen-binding capacity of the bispecific antibodies.

[0236] Example 8 Bisspecific Antibody Binding Activity to Fc Receptors

[0237] The affinity of the bispecific antibody purified in Example 6 for binding to human FcγRI receptor and human FcRn was determined using biofilm thin layer interferometry (BLI) using a Fortebio Octet Red 96 instrument and the equilibrium dissociation constant (KD) was calculated.

[0238] A column of HIS1K sensors (Fortebio, Cat 18-5120) was immersed in standard buffer (1× PBS, pH 7.4 with 0.1% BSA, 0.02% Tween-20). After equilibration in standard buffer, 5 μg / ml of recombinant human FcγRI protein (ACRO Biosystems, Cat FCA-H52H1) was immobilized on the sensors. The sensors were then bound to 100 nM antibody and dissociated in standard buffer. The instrument run steps were: Baseline 1 (60 s), Loading (~15 s, 0.5 nm), Baseline 2 (60 s), Association (60 s), and Dissociation (120 s). The instrument was operated at 1000 rpm and 30°C.

[0239] A column of SA sensors (Fortebio, Cat 18-5019) was immersed in pH 6.0 buffer (1× PBS, pH 6.0, with 0.1% BSA and 0.02% Tween-20). After the sensors were equilibrated in pH 6.0 buffer, 200 μl of pH 6.0 buffer, 100 nM antibody, and human FcRn were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a Fortebio Octet Red96 assay, with sensor positions selected based on sample placement. After equilibration in pH 6.0 buffer, 2 μg / ml of recombinant human FcRn protein (ACROBiosystems, Cat FCM-H8286) was immobilized on the sensors. The sensors were then bound to 100 nM antibody and dissociated in pH 6.0 buffer. Instrument operation steps: Baseline1 (60s), Loading (~100s, 3.0nm), Baseline2 (60s), Association (60s) and Dissociation (60s), the rotation speed is 1000rpm, and the temperature is 30℃.

[0240] The results of the Fortebio test are shown in Figure 8. The affinity of the control antibody IgG1 to human FcγRI is 6.33x 10 -9 M; The affinity of the combination of KIH, M1, M2 and M3 mutants to human FcγRI is 1.01x 10 -8 M to 1.03x 10 -8 The affinity of the control antibody IgG1 to human FcRn is 1.41x 10 -8 The affinity of the combination of KIH, M1, M2 and M3 mutants to FcRn was slightly lower, at 1.44 x 10 -8 M to 1.85x 10 -8The affinity differences between the M1, M2, and M3 mutants were within the error range of the instrument. Therefore, the Fc mutant combinations obtained in this study essentially maintained binding to both human FcRn and human FcγRI. Furthermore, the affinity values ​​of the M1, M2, and M3 mutant combinations were very similar to those of the KIH control.

[0241] Example 9: Accelerated stability test of dual antibodies

[0242] The purified bispecific antibody from Example 6 was diluted to 1 mg / ml using a 20 mM histidine buffer and filtered through a 0.22 μm PES filter. The sample was placed in a 42°C oven and sampled after 7 and 14 days for purity analysis using SEC-HPLC and SDS-PAGE.

[0243] The results of SEC-HPLC analysis are shown in Table 9. After storage at 42°C for 7 and 14 days, the heterodimer purity of each mutant combination decreased only slightly compared to day 0. The purity of the KIH, M1, M2, and M3 mutant combinations decreased by 0.21%, 0.73%, 0.47%, and 0.57%, respectively, on day 7; and by 1.43%, 1.50%, 1.46%, and 0.84%, respectively, on day 14. These results demonstrate that the newly designed Fc mutant combination exhibits excellent stability under high-temperature storage conditions.

[0244] The results of SDS-PAGE analysis also showed that after the KIH, M1, M2 and M3 mutant combinations were stored at 42°C for 14 days, the ratios of protein bands of different molecular weights remained basically unchanged compared to those at 0 days.

[0245] Table 9 SEC-HPLC analysis of storage stability of bispecific antibodies in accelerated experiments

[0246] discuss

[0247] By analyzing the reasons for the reduced thermal stability of the handle-hole model, we selected amino acids near the CH3 contact interface of the Fc fragment as mutation targets to investigate the effects of different mutation combinations on the production performance, physicochemical properties, and biological functions of Fc-based IgG-like mono / multispecific antibodies.

[0248] Without wishing to be bound by theory, it is proposed that in wild-type IgG1 Fc, amino acid L351 in one chain makes direct contacts with L351, P352, P353, S354, and T366 in the opposing side chain. In contrast, in the KIH structure, L351 in the hole chain makes direct contacts with L351, P352, P353, S354, and T366W in the handle chain, but the contact distance is increased, and the interaction is expected to weaken. Mutating hole L351, such as to a tyrosine with a larger side chain (L351Y), is hypothesized to bring the hole L351 residue into closer contact with handle amino acids P352, P353, S354, and T366W, thereby creating stronger hydrophobic interactions (see Figure 1C) and creating new contacts with other handle amino acids, such as the formation of a new hydrogen bond network between the hydroxyl group (OH) of L351Y and T366W and E357.

[0249] Based on the hole chain L351 mutation, a combination of handle chain K409D and hole chain D399R mutations was further introduced. Without wishing to be bound by theory, it is speculated that this combination of mutations could result in a pair of salt bridges similar to those in the IgG1 Fc. Furthermore, hole chain D399R would create new contacts with handle chain amino acids K392, T411, and T366W, while the handle chain K409D mutation would maintain a direct contact with hole chain F405 (see Figure 1D).

[0250] The experimental results of the above examples show that the combination of the above mutations in the KIH structure, i.e., the hole chain L351Y or the hole chain L351Y+handle chain K409D-D399R mutation, can give a good promoting effect on the production and physicochemical properties of the heterodimeric Fc scaffold and the single / multi-specific proteins (especially bispecific antibodies) containing it. After the Fc heterodimer scaffold protein containing the mutation combination is expressed in cells and purified by affinity chromatography, the ratio of Fc heterodimers produced can reach more than 90%, which is higher than the control Fc protein containing only the corresponding knob-into-hole mutation; and after further purification by ion exchange column, the purity can reach more than 98%. In addition, compared with the control Fc protein, the purified Fc heterodimer scaffold protein has better thermal stability, wherein the CH3 domain Tm value determined by the DSC method can reach more than 74°C.

[0251] Furthermore, the experimental results of the above examples also show that the combination of the above mutations in the KIH structure, i.e., the hole chain L351Y or the hole chain L351Y + handle chain K409D-D399R mutations, has basically no effect on the antigen binding function and FcRn and FcγR receptor binding function of the heterodimeric Fc scaffold and the single / multispecific proteins (especially bispecific antibodies) containing it.

[0252] Based on these results, it can be concluded that introducing the hole L351Y mutation or the combination of the hole L351Y-D399R and the handle K409D mutation on the basis of KIH will be beneficial to the production and stability of heterodimeric target antibodies.

[0253] Sequence Listing

Claims

1. A heterodimeric Fc scaffold comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first Fc region, and the second polypeptide comprises a second Fc region, wherein the first Fc region comprises a first CH3 domain, and the second Fc region comprises a second CH3 domain, in, Relative to the wild-type CH3 domain, the first Fc region and the second Fc region respectively comprise a knob mutation and a hole mutation of a KIH mutation in their CH3 domains, and wherein, (a) the Fc region comprising the hole mutation further comprises an L351Y mutation; or (b) the Fc region comprising the hole mutation further comprises L351Y and D339R mutations, and the Fc region comprising the knob mutation further comprises K409D mutation, wherein the first polypeptide and the second polypeptide pair and heterodimerize via the first and second Fc regions to form the Fc scaffold, The amino acid residues are numbered according to the EU index in Kabat.

2. The heterodimeric Fc scaffold of claim 1, wherein The first and second Fc regions comprise CH2 and CH3 domains, or comprise a hinge region, CH2 and CH3 domains.

3. The heterodimeric Fc scaffold of any one of claims 1 to 2, wherein The first and second Fc regions are of IgG type, such as IgG1, IgG2, IgG3 or IgG4 subtype. Preferably, the first and second Fc regions are of human IgG1 subtype.

4. The heterodimeric Fc scaffold of any one of claims 1 to 3, wherein the knob mutation is T366W; and the hole mutation is Y407V or T366S-L368A-Y407V, Preferably, the first Fc region comprises T366W, and the second Fc region comprises T366S-L368A-Y407V-L351Y; or The first Fc region comprises T366W-K409D, and the second Fc region comprises T366S-L368A-Y407V-L351Y-D399R.

5. The heterodimeric Fc scaffold of any one of claims 1 to 4, wherein (a) relative to the wild-type CH3 domain, the first and second CH3 domains respectively further comprise 0-3 amino acid residue changes, preferably, the wild-type CH3 domain is a natural human IgG1 immunoglobulin CH3 domain, more preferably comprising the amino acid sequence of SEQ ID NO: 19 or 20; (b) the first and second Fc regions comprise a native human IgG1 immunoglobulin CH2 domain, or a CH2 domain comprising the amino acid sequence of SEQ ID NO: 21 or having 1-5 amino acid residues altered therefrom; (c) the Fc region comprising a knob mutation comprises the amino acid sequence of SEQ ID NO: 1 or 2, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto; (d) the Fc region comprising the hole mutation comprises the amino acid sequence of SEQ ID NO: 7 or 8, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, and / or (e) The Fc scaffold does not form a non-native disulfide bond between the first Fc region and the second Fc region.

6. The heterodimeric Fc scaffold of any one of claims 1-5, wherein the Fc scaffold has increased thermal stability compared to a corresponding control Fc scaffold comprising only the KIH mutation, and preferably, the CH3 domain of the Fc scaffold has a Tm value of about 70°C or higher as determined by DSC.

7. A CH3 heterodimer comprising a heterodimerized first immunoglobulin CH3 domain and a second immunoglobulin CH3 domain, wherein: Relative to the wild-type CH3 domain, the first CH3 domain and the second CH3 domain respectively comprise a knob mutation and a hole mutation in the KIH mutation, and wherein, (a) the CH3 domain comprising the hole mutation further comprises an L351Y mutation; or (b) A CH3 domain comprising a hole mutation further comprising L351Y and D339R mutations and a CH3 domain comprising a knob mutation It also contains the K409D mutation, The amino acid residues are numbered according to the EU index in Kabat. Preferably, the first and second CH3 domains are of IgG type, such as IgG1, IgG2, IgG3 or IgG4 subtype, Preferably, the wild-type CH3 domain is a natural human IgG1 immunoglobulin CH3 domain; More preferably, relative to the wild-type CH3 domain, the first and second CH3 domains further comprise 0-3 amino acid residue changes respectively.

8. A heteromultimeric protein comprising the heterodimeric Fc scaffold of any one of claims 1 to 6 or the CH3 heterodimer of claim 7.

9. A binding protein comprising the heterodimeric Fc scaffold of any one of claims 1 to 6 and at least one target binding domain linked thereto.

10. The binding protein of claim 9, wherein the at least one target binding domain is independently selected from the group consisting of: a ligand, a receptor binding domain of a ligand, a ligand binding domain of a receptor, and an antigen binding domain of an antibody.

11. The binding protein of any one of claims 9-10, wherein the binding protein is monospecific or bispecific.

12. The binding protein of any one of claims 9-11, wherein the binding protein comprises at least one target binding domain independently selected from Fv, scFv, Fab, scFab, crossFab, VHH and a ligand, preferably 1-6, more preferably 1, 2 or 3 of the target binding domains.

13. The binding protein of any one of claims 9-12, wherein said binding protein comprises only one of said heterodimeric Fc scaffolds.

14. The binding protein of any one of claims 9 to 13, wherein the binding protein has an IgG configuration or an IgG-like configuration, Preferably, the binding protein has a configuration selected from the following: scFv-Fc / scFv-Fc, Fab-Fc / Fab-Fc, VHH-Fc / VHH-Fc, VHH-Fc / Fab-Fc, VHH-Fc / scFv-Fc, VHH-Fc / Fc, scFv-Fc / Fc, Fab-Fc / Fc, ligand-Fc / ligand-Fc, ligand-Fc / scFv-Fc, ligand-Fc / VHH-Fc, ligand-Fc / Fab-Fc, ligand-Fc / Fc.

15. The binding protein of any one of claims 9 to 14, wherein the binding protein is bispecific and comprises: (a) two different Fab domains connected to the N-termini of the first and second Fc regions of the heterodimeric Fc scaffold, respectively, wherein the Fab domains have the same or different VLCL light chains; or (b) a VHH domain and a Fab domain connected to the N-termini of the first and second Fc regions of the heterodimeric Fc scaffold, respectively.

16. The binding protein of any one of claims 9 to 15, having one or more properties selected from the group consisting of: - Thermal stability, among which The CH3 domain has a Tm value greater than or equal to 74°C as determined by DSC; - 90% purity or higher; - The binding affinity to the FcRn receptor is not less than 80% of the binding affinity of the natural IgG1 monoclonal antibody to the FcRn receptor, preferably not less than 85%, 90%, or 95%; - The binding affinity to FcγR1 receptor is not less than 50% of the binding affinity of natural IgG1 monoclonal antibody to FcγR1 receptor; - Storage stability, wherein the purity change is less than 2% after storage at 42°C for 2 weeks as determined by SEC-HPLC.

17. A polynucleotide encoding the heterodimeric Fc scaffold of any one of claims 1 to 6, the CH3 heterodimer of claim 7, the heteromultimeric protein of claim 8, or the binding protein of any one of claims 9 to 16.

18. A vector or host cell comprising the polynucleotide of claim 17.

19. A composition comprising the heteromultimeric protein of claim 8 or the binding protein of any one of claims 9-16.

20. A conjugate comprising a therapeutic agent or a diagnostic agent conjugated to the heteromultimeric protein of claim 8 or the binding protein of any one of claims 9-16.

21. Use of the composition of claim 19 or the conjugate of claim 20 as a medicament or in the preparation of a medicament, preferably for cancer treatment.

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