Antibody-like molecules containing heterodimers of human CD1b (differentiation antigen group 1) protein

Abstract

The present invention relates to the field of biotechnology, specifically to an antibody-like molecule comprising a heterodimer of the membrane-proximal domain of the human CD1b (differentiation antigen group 1) protein, and to a method for producing the antibody-like molecule. The present invention further relates to a nucleic acid encoding the antibody-like molecule, an expression vector, a host cell for producing the antibody-like molecule, and a method for producing the cell.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically to antibody-like molecules comprising a heterodimer of the membrane-proximal domain of human CD1b (cluster of differentiation 1) protein, and to methods for producing said antibody-like molecules. The present invention further relates to nucleic acids encoding said antibody-like molecules, expression vectors, host cells for producing said antibody-like molecules and methods for producing said cells.

Background Art

[0002] Monoclonal antibodies in the form of chimeric, humanized or fully human molecules have proven to be useful as effective pharmaceuticals for treating multiple disorders and diseases. Natural human antibody molecules are represented by a heterotetramer composed of two light chains and two heavy chains: the heavy chains form homodimers and each heavy chain forms a heterodimer with a cognate light chain. Conventional monoclonal antibodies in the form of complete molecules are composed of bivalent ("2-arm") heterotetramers of heavy and light chains.

[0003] Diseases are often caused as a result of several pathologies and are associated with numerous complications. Bispecific and multispecific antibodies can bind to two or more distinct antigens per antibody molecule, and thereby neutralize them. The potential to significantly improve the therapeutic properties (and value) of pharmaceuticals, compared to single-specific monoclonal antibodies, has made bispecific and multispecific antibodies an active area of research. Over the past 20 years, the literature has described numerous solutions for engineered versions of bispecific antibodies, as described in Brinkmann, U and RE Kontermann, 2017, The Making of Bispecific Antibodies, MAbs; 209 Feb / Mar; 9(2): 182-212, doi: 10.1080 / 19420862.2016.1268307.

[0004] As mentioned above, there are numerous approaches to creating molecules that have combined antigen-binding domains, that is, molecules with different antigen-binding domains. However, each of these methods has its drawbacks.

[0005] Chemical crosslinking is a time-consuming process because the corresponding portion needs to be purified from homodimers and other undesirable by-products. In addition, the chemical modification step may alter the integrity of the protein, potentially compromising its stability. Thus, the above method is usually ineffective and may even lead to a loss of antibody activity.

[0006] Cell fusion-based methods (e.g., hybridoma production) involve any assembly of two heavy chains and two light chains, resulting in 10 possible combinations of antibodies. The target heteromultimeric antibody is only a small fraction of the antibodies produced in this manner. Isolating the target heteromultimeric protein significantly reduces product yield and increases manufacturing costs.

[0007] Recombinant DNA techniques are used to produce various heteromultimeric antibodies, such as single-chain Fv fragments and diabodies that do not contain Fc fragments. The main drawback of this type of antibody molecule is the absence of an Fc domain, which results in antibodies that cannot induce effector functions (e.g., complement activation, binding to Fc receptors, etc.). Thus, bispecific or multispecific antibodies containing a functional Fc domain are needed.

[0008] Recombinant DNA techniques are also used to design bispecific or multispecific antibodies coupled using knob-in-hole techniques (see, for example, International Patent Publications WO9627011 and WO9850431). One factor limiting the use of the above methods is the fact that, when expressed in single cells, the light chains of the first two antibodies must be identical to prevent mispairing and formation of undesirable and / or inactive molecules.

[0009] The purity of the product of bispecific or multispecific antibodies depends on the following two factors: a) A heterodimer assembly of two distinct heavy chains co-expressed in cells, and b) Precise pairing of two distinct light chains with a cognate heavy chain.

[0010] The "knob-into-hole" technique for designing bispecific or multispecific antibodies solves the problem of precise heterodimer assembly of two distinct heavy chains co-expressed in cells. However, using only the knob-into-hole technique for designing bispecific or multispecific antibodies results in a yield of only about 25% for precisely assembled bispecific or multispecific products because the problem of precise pairing of two distinct light chains to their homologous heavy chains remains unresolved.

[0011] The problem of the precise pairing of two distinct light chains with their corresponding heavy chains is solved in the following various ways: 1. Use of the same light chain in the first and second antigen-binding moieties of the antibody (Van Blarcom T et al., Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies, MAbs. 2018 Feb / Mar;10(2):256~268.doi:10.1080 / 19420862.2017.1406570).

[0012] The drawback of the above solution is its non-universality, because selecting a light chain appropriate for both binding valencies can be problematic. Furthermore, in the case of amino acid substitutions in the light chain to optimize the properties of the antigen-binding fragment, such substitutions will affect both binding valencies. Moreover, the binding of the antibody to the second antigen may be hindered.

[0013] 2. Use of a single-chain format, i.e., a format in which the light and heavy chains of antigen-binding fragments specific to the first antigen are interconnected via linkers of several amino acids. This format has technical drawbacks because it uses a linker either to fuse the antibody core (IgA, IgD, IgE, IgG, or IgM) to a further binding protein (e.g., scFv or scFab), or to fuse, for example, the variable domains of the light and heavy chains (VH and VL) within scFv or the light chain (VL-CK (or CL)) with VH-CH1 within scFab. Linkers can cause problems in therapeutic settings. In fact, such exogenous peptides may elicit an immune response to the linker itself or to the binding region between the protein and the linker. Furthermore, the flexibility and mobility of these peptides make them more susceptible to cleavage by proteolysis, which can result in lower antibody stability, aggregation, and increased immunogenicity.

[0014] 3. Modification of the CH1-CK domain in bispecific or multispecific antibody that enables the elimination of inaccurate association of light chains by altering the interaction interface in bispecific or multispecific antibody expression techniques. For example, International Patent Publication WO2017059551 provides various amino acid substitutions in CH1 and / or CK that promote favorable pairing between a desired heavy chain and a desired light chain. [Overview of the Initiative] [Problems that the invention aims to solve]

[0015] Despite the various bispecific or multispecific antibody expression techniques described above, there remains a need in the art for improving the purity of bispecific or multispecific antibody products, and for scalable production solutions for accurately assembled bispecific or multispecific antibodies. [Means for solving the problem]

[0016] The novel format of an antibody-like molecule containing a heterodimer of the membrane-proximal domain of the CD1b (differentiation antigen group 1) protein, and the method for producing said antibody-like molecule, developed by the authors of this invention, remarkably enables the production of high-yield products having a precise heterodimer assembly of two distinct heavy chains co-expressed intracellularly and precise pairing of two distinct light chains to a congeneral heavy chain.

[0017] The novel format of an antibody-like molecule containing a heterodimer of the membrane-proximal domain of the CD1b protein, developed by the authors of this invention, and the method for producing said antibody-like molecule, remarkably enable the production of precisely assembled products of high-purity antibody-like molecules.

[0018] As a result, the above findings provide a scalable manufacturing solution for reducing production costs and producing properly assembled antibody-like molecules. [Brief explanation of the drawing]

[0019] [Figure 1] This is an electrophoresis map of an antibody-like molecule sample following purification from the culture medium. 7.5% PAAG, non-reducing conditions. M - Precision Plus Protein™ Dual Color Standards (BIO-RAD) standard marker. The molecular weight of the corresponding lane is shown as kDa in the column on the left side of the gel; 1 - 08-001; 2 - 08-002; 3 - 08-003; 4 - 08-004; 5 - 08-005; 6 - 08-006. [Figure 2] This is an electrophoresis image of an antibody-like molecule sample following purification from the culture medium. 12% PAAG, reducing conditions.

[0020] M - Standard marker for Precision Plus Protein (trademark) Dual Color Standards (BIO-RAD). The molecular weight of the corresponding lane is shown as kDa in the column on the left side of the gel; 1 - 08-001; 2 - 08-002; 3 - 08-003; 4 - 08-004; 5 - 08-005; 6 - 08-006. [Figure 3] Electrophoretogram of a sample of an antibody-like molecule before cleavage by GingisKHAN protease and the subsequent one. 7.5% PAAG, non-reducing conditions. [[ID=…]]

[0021] M - Standard marker of Precision Plus Protein™ Dual Color Standards (BIO-RAD). The molecular weights of the corresponding lanes are shown as kDa in the column on the left side of the gel; 1 - 08-001 Intact sample; 2 - 08-001 Following proteolysis; 3 - 08-002 Intact sample; 4 - 08-002 Following proteolysis; 5 - 08-003 Intact sample; 6 - 08-003 Following proteolysis. [Figure 4] Electrophoretogram of a sample of an antibody-like molecule before cleavage by GingisKHAN protease and the subsequent one. 7.5% PAAG, non-reducing conditions.

[0022] M - Standard marker of Precision Plus Protein™ Dual Color Standards (BIO-RAD). The molecular weights of the corresponding lanes are shown as kDa in the column on the left side of the gel; 1 - 08-004 Intact sample;[[ID=…]] 2 - 08-004 Following proteolysis; 3 - 08-005 Intact sample; 4 - 08-005 Following proteolysis; 5 - 08-006 Intact sample; Note: Some of the consecutive tags like

[0021] etc. were replaced with

[0021] (… in the translation) as they were not translated in the original instruction. If these are meant to be translated in some specific way, more information would be needed to provide a more accurate translation for them.6 - 08-006 Continues with protein degradation. [Figure 5] Electrophoretic maps of bispecific and monospecific antibody-like molecules following cleavage by GingisKHAN protease. 7.5% PAAG, non-reducing conditions.

[0023] M - Standard marker for Precision Plus Protein (trademark) Dual Color Standards (BIO-RAD). The molecular weight of the corresponding lane is shown as kDa in the column on the left side of the gel; 1 - A bispecific antibody-like molecule comprising the variable domains of the antibody prorugolimab and the antibody ocrelizumab, and a dimerization unit of the membrane-proximal domain of the CD1b protein; 2. A bispecific antibody-like molecule comprising the variable domains of the antibody prorugolimab and anti-CSF1R, and a dimerization unit of the membrane-proximal domain of the CD1b protein.

[0024] 3 - Antibody prorugolimab; 4 - A monospecific antibody-like molecule containing a dimerization unit based on the membrane-proximal domain of the CD1b protein and a variable fragment of the antibody prorugolimab; 5 - A monospecific antibody containing the variable domain of the antibody ocrelizumab. [Modes for carrying out the invention]

[0025] Definitions and general methods Unless otherwise defined herein, all technical and scientific terms used in connection with the present invention shall have the same meaning as those commonly understood by those skilled in the art.

[0026] Furthermore, unless otherwise required by context, singular terms shall encompass plural terms, and plural terms shall encompass singular terms. Generally, the classifications and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, and medical and pharmaceutical chemistry, as well as the hybridization and chemistry of proteins and nucleic acids described herein, are well known to those skilled in the art and are widely used in the art. Enzymatic reactions and purification methods are carried out as is common in the art, or in accordance with the manufacturer's guidelines as described herein.

[0027] In this specification, the term "KD" refers to the affinity constant (or equilibrium constant) calculated from the ratio of Kd to Ka (i.e., Kd / Ka), and is expressed as molar concentration (M).

[0028] "Binding affinity" typically refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise noted, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its binding partner Y can typically be expressed by an affinity constant (KD). Preferred Kd values ​​are about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM, or less. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies typically bind slowly to antigens and readily dissociate, while high-affinity antibodies typically bind more quickly to antigens and remain bound for longer periods. Various methods for measuring binding affinity are known in the art, and any one of these methods may be used for the purposes of the present invention.

[0029] The terms "Kd," "koff," or "kdis" refer to the dissociation rate constant of a particular interaction between a binding molecule and an antigen. The dissociation rate constant koff can be measured using biolayer interferometry, for example, using the Octet® system.

[0030] The terms "Ka," "kon," or "on-rate" refer to the bond rate constant. "R 2 The term "coefficient of determination" refers to the coefficient of determination.

[0031] As used herein and in the following claims, unless otherwise specified by context, the words “include” and “comprise,” or their variations such as “includes,” “including,” “comprises,” or “comprising,” are understood to mean the inclusion of the integer or group of integers described, but not the exclusion of any other integer or group of integers.

[0032] Antibody-like molecules The present invention relates to antibody-like molecules that specifically bind to first and second targets. The antibody-like molecule according to the present invention is a monoclonal antibody-like molecule.

[0033] The term "monoclonal antibody-like molecule" refers to an antibody-like molecule synthesized and secreted by cells of a separate clonal population. The antibody-like molecule according to the present invention is a recombinant antibody-like molecule.

[0034] The term "recombinant antibody-like molecule" refers to an antibody-like molecule expressed in a cell or cell line containing a nucleotide sequence encoding an antibody-like molecule, where the nucleotide sequence is not naturally associated with the cell. The antibody-like molecule according to the present invention is an isolated antibody-like molecule.

[0035] The term “isolated” as used to describe the various antibody-like molecules in this specification refers to antibody-like molecules that have been identified, isolated, and / or regenerated from cells or cell cultures on which they are expressed. Natural environmental impurities (contaminating components) are materials that typically interfere with the diagnostic or therapeutic use of polypeptides and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated polypeptides are typically prepared by at least one purification step.

[0036] In one aspect, the present invention relates to an antibody-like molecule that specifically binds to a first and a second target, wherein the antibody-like molecule is: (i)a) The light chain constant domain of the human CD1b protein, which is β2 microglobulin (β2M); and b) The first heavy chain constant domain, which is the α3 membrane proximal domain of the human CD1b protein; or (ii) a) The light chain constant domain, which is the α3 membrane proximal domain of the human CD1b protein; and b) The first heavy chain constant domain of the human CD1b protein, which is β2 microglobulin (β2M) Includes; Here, the β2 microglobulin (β2M) of the human CD1b protein and the α3 membrane proximal domain of the human CD1b protein form a heterodimer between them, which is stabilized by a disulfide bond.

[0037] The α3 domain of the human CD1b protein (the α3 membrane-proximal domain of the human CD1b protein) and the β2 microglobulin of the human CD1b protein are membrane-proximal domains of the human CD1b protein.

[0038] In one aspect, the present invention relates to an antibody-like molecule that specifically binds to a first and a second target, wherein the antibody-like molecule is: (i)a) The light chain constant domain of human CD1b protein, which is β2 microglobulin (β2M) having the amino acid sequence of Sequence ID No. 1; and b) The first heavy chain constant domain, which is the α3 membrane proximal domain of the human CD1b protein, having the amino acid sequence of Sequence ID No. 2; or (ii)a) The light chain constant domain, which is the α3 membrane proximal domain of the human CD1b protein, having the amino acid sequence of Sequence ID No. 2; and b) The first heavy chain constant domain of human CD1b protein, which is β2 microglobulin (β2M) having the amino acid sequence of Sequence ID No. 1. Includes; Here, the β2 microglobulin (β2M) of the human CD1b protein and the α3 membrane proximal domain of the human CD1b protein form a heterodimer between them, which is stabilized by a disulfide bond.

[0039] In one aspect, the present invention relates to an antibody-like molecule that specifically binds to a first and a second target, wherein the antibody-like molecule: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to the first target and includes the following: a) The light chain of the first antigen-binding fragment, wherein the light chain of the first antigen-binding fragment comprises a light chain variable domain and a light chain constant domain; And, b) The heavy chain of the first antigen-binding fragment, wherein the heavy chain of the first antigen-binding fragment comprises a variable heavy chain domain of the antibody and a constant heavy chain domain of the antibody, and the constant heavy chain domain comprises a first (CH1) constant heavy chain domain and an Fc fragment monomer, wherein the Fc fragment monomer comprises a second (CH2) constant heavy chain domain and a third (CH3) constant heavy chain domain; and, 2) The second antigen-binding fragment specifically binds to the second target and comprises either (i) or (ii) below: (i) a) A light chain of a second antigen-binding fragment, wherein the light chain of the second antigen-binding fragment comprises a light chain variable domain and a constant domain which is β2 microglobulin (β2M) of the human CD1b (differentiation antigen group 1) protein having the amino acid sequence of Sequence ID No. 1; And, b) The heavy chain of the second antigen-binding fragment, which comprises a heavy chain variable domain, a constant domain which is the α3 membrane proximal domain of the human CD1b protein having the amino acid sequence of SEQ ID NO: 2, and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; or, (ii) a) The light chain of the second antigen-binding fragment, wherein the light chain of the second antigen-binding fragment comprises a light chain variable domain and a constant domain which is the α3 membrane proximal domain of the human CD1b protein having the amino acid sequence of SEQ ID NO: 2; And, b) The heavy chain of the second antigen-binding fragment, wherein the heavy chain of the second antigen-binding fragment comprises a heavy chain variable domain, a constant domain which is β2 microglobulin (β2M) of the human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; Here, the β2 microglobulin (β2M) of the human CD1b protein, which has the amino acid sequence of SEQ ID NO: 1, and the α3 membrane proximal domain of the human CD1b protein, which has the amino acid sequence of SEQ ID NO: 2, form a heterodimer between them, stabilized by a disulfide bond.

[0040] In the antibody-like molecule described above, which specifically binds to the first and second targets, the variable domain and the constant domain are arranged in the heavy and light chains in the following order: The light chain of the first antigen-binding fragment: 1) Light chain variable domain, 2) Light chain constant domain; Heavy chain of the first antigen-binding fragment: 1) Heavy chain variable domain, 2) The first (CH1) heavy chain constant domain, 3) The second (CH2) heavy chain constant domain, and 4) The third (CH3) heavy chain constant domain; The light chain of the second antigen-binding fragment: 1) Light chain variable domain, 2) The first membrane-proximal domain of the human CD1b protein; The heavy chain of the second antigen-binding fragment: 1) Heavy chain variable domain, 2) Second membrane-proximal domain of human CD1b protein 3) The second (CH2) heavy chain constant domain, and 4) The third (CH3) heavy chain constant domain.

[0041] The antibody-like molecule of the present invention refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The first light chain consists of a light chain variable region (abbreviated as VL1 in this specification) and a light chain constant region.

[0042] Preferably, the first light chain is a kappa (κ) light chain, and the constant domain CL is preferably C-kappa (κ). The first heavy chain comprises a heavy chain variable region (abbreviated as VH1 in this specification) and a heavy chain constant region containing CH1-CH2-CH3. The second light chain consists of a light chain variable region (abbreviated as VL2 in this specification) and a light chain constant region represented by the first membrane proximal domain of the human CD1b protein. The second heavy chain comprises a heavy chain variable region (abbreviated as VH2 in this specification) and a heavy chain constant region containing the second membrane proximal domain of the human CD1b protein and CH2-CH3.

[0043] The antibody-like molecule according to the present invention may be an antibody-like molecule of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, preferably IgG1).

[0044] The VL and VH regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are separated from more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0045] The constant region of an antibody-like molecule can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0046] As used herein, the term "antigen-binding fragment" refers to one or more fragments of an antibody-like molecule that possess the ability to specifically bind to an antigen. As used in this application, “Kabat numbering scheme” or “Kabat-based numbering” refers to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions of the heavy and light chains of antibody-like molecules (Kabat et al. Ann. NYAcad. Sci., 190:382~93 (1971); Kabat et al. Sequences of Proteins of Immunological Interest, 5th edition, USD Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0047] The antibody-like molecule of the present invention that "specifically binds" to a target antigen or target refers to an antibody-like molecule that binds to the antigen or target with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent targeting a protein, cell, or tissue expressing the antigen, and that cross-reacts only slightly with other proteins.

[0048] The term "specifically binds to" an epitope on a particular polypeptide or a particular target polypeptide can be illustrated by the example of molecules having a Kd of at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM or less to a target.

[0049] In one embodiment, the term “specific binding” refers to a binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or epitope on a polypeptide.

[0050] The crystallizable region of an immunoglobulin fragment ("Fc region, Fc") is the terminal region of an immunoglobulin molecule that interacts with cell surface Fc receptors as well as some proteins of the complement system. This property allows antibodies or antibody-like molecules according to the present invention to activate the immune system. In the IgG, IgA, and IgD isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of two heavy chains, respectively; in the IgM and IgE isotypes, Fc contains three heavy chain constant domains (CH2, CH3, and CH4 domains) in their respective polypeptide chains.

[0051] The term "Fc fragment monomer" is understood to mean the Fc region derived from the second and third constant domains of either of the two heavy chains (for each isotype of IgG, IgA, and IgD); for the isotypes of IgM and IgE, the Fc monomer contains three constant domains (the CH2, CH3, and CH4 domains) of one of the two heavy chains.

[0052] CD1 (differentiation antigen group 1) refers to a differentiation antigen group 1 molecule, a component of the immune system, located on the surface of various antigen-presenting cells such as dendritic cells, macrophages, and other cells. Similar to MHC classes I and II, CD1 presents antigens for recognition by T cells through interaction with T cell receptors. Unlike MHC classes I and II, CD1 proteins present lipids and their derivatives rather than peptides.

[0053] Multiple CD1 variants found in humans are classified into five groups: CD1a, CD1b, CD1c, CD1d, and CD1e. These differ in the structure of their antigen-binding fragments and, consequently, in their specificity for different lipid structures. Unlike the other groups of proteins, the CD1e protein is not expressed on the cell surface but is soluble and involved in lipid transport (Kaczmarek, R., Pasciak, M., Szymczak-Kulus, K., & Czerwinski, M. (2017). CD1: A Singed Cat of the Three Antigen Presentation Systems. Archivum Immunologiae et Therapiae Experimentalis, 65(3), 201-214).

[0054] CD1 molecules are structurally similar to MHC class I molecules. In a similar manner, a single CD1 molecule is a non-covalent complex composed of two polypeptide chains: a polymorphic α-chain (sometimes called a heavy chain) and a smaller chain called β2-microglobulin (also known as a light chain), which is generally not polymorphic.

[0055] The α chain forms an antigen-binding region containing the α1 and α2 domains. The α2 domain is followed by the α3 domain located at the C-terminus of the extracellular portion of the CD1 α chain, and together with β2 microglobulin, forms a heterodimer non-covalent complex. This heterodimer non-covalent complex, composed of the α3 domain of CD1 and β2 microglobulin, is referred to in the specification of this invention as the heterodimer of the membrane-proximal domain of the CD1b protein.

[0056] The membrane-proximal domain of the human CD1b protein is understood to refer to the α3 domain and the β2 microglobulin of the human CD1b protein. The β2M of human CD1b protein in the antibody-like molecule according to the present invention is the β2M of wild-type human CD1b protein having the amino acid sequence of SEQ ID NO: 1.

[0057] The α3 membrane proximal domain of the human CD1b protein in the antibody-like molecule according to the present invention is the α3 membrane proximal domain of the human CD1b protein having the amino acid sequence of SEQ ID NO: 2, and differs from the α3 membrane proximal domain of the wild-type human CD1b protein having the amino acid sequence of SEQ ID NO: 3 in that it has S12K and N59D mutations and GSC elongation at the C-terminus.

[0058] In some aspects of the present invention, the antibody-like molecule comprises a CH3 domain of one heavy chain and a CH3 domain of the other heavy chain that are in contact with each other via a modified surface to form the antibody-like molecule, wherein the modification in the CH3 domains of the heavy chains is a substitution to result in heterodimerization.

[0059] In some aspects of the present invention, the antibody-like molecule is: a) One heavy chain's CH3 domain is modified such that, on the surface of the CH3 domain of one heavy chain, it comes into contact with the surface of the CH3 domain of the other heavy chain in the antibody-like molecule, and an amino acid residue is substituted with an amino acid residue having a larger side chain volume, resulting in the formation of a knob on the surface of the CH3 domain of the other heavy chain, which can fit into a hole on the surface of the CH3 domain of the other heavy chain. On the other hand, the CH3 domain of the heavy chain, b) The CH3 domain of the other heavy chain, which is modified such that, on the surface of the CH3 domain of the second heavy chain, it is in contact with the surface of the CH3 domain of the first heavy chain in the antibody-like molecule, and an amino acid residue is substituted with an amino acid residue having a smaller side-chain volume, resulting in the formation of a hole on the surface of the CH3 domain of the second heavy chain that can fit onto a knob on the interface of the CH3 domain of the first heavy chain. The CH3 domain of the other heavy chain and Includes; Here, the amino acid residue having a larger side chain volume is selected from the group including arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Furthermore, the amino acid residue having a smaller side chain volume is selected from the group including alanine (A), serine (S), threonine (T), and valine (V).

[0060] In some aspects of the present invention, the antibody-like molecule comprises a constant domain of the first light chain of an antibody, selected from CK or CL. In some aspects of the present invention, the antibody-like molecule comprises the CH3 domain of an antibody, which is modified by introducing cysteine ​​(C) as an amino acid to the corresponding position of each CH3 domain so that a disulfide crosslink can be formed between both CH3 domains.

[0061] In some aspects of the present invention, the antibody-like molecule comprises a CH3 domain of one heavy chain modified to form a knob and a CH3 domain of the other heavy chain modified to form a hole, or vice versa.

[0062] "Knob-into-hole" ("knob-into-hole" type interaction) is an approach that can avoid problems associated with mispairing byproducts. This technique forces pairing of two different antibody heavy chains of an antibody or antibody-like molecule by introducing mutations into the CH3 domain to modify the contact interface. On one chain, a bulky amino acid is replaced by an amino acid with a shorter side chain, creating a "hole." Conversely, an amino acid with a larger side chain is introduced into the CH3 domain of the other chain, creating a "knob." Co-expression of these two heavy chains has produced high-yield heterodimerization ("knob-hole") in contrast to homodimerization ("hole-hole" or "knob-knob") (see, for example, International Patent Publications WO9627011 and WO9850431).

[0063] In some aspects of the present invention, the antibody-like molecule comprises a CH3 domain of one heavy chain having the amino acid substitution S354C / T366W according to the EU amino acid numbering scheme for antibodies, and a CH3 domain of the other heavy chain having the amino acid substitution Y349C / T366S / L368A / Y407V according to the EU amino acid numbering scheme for antibodies.

[0064] In some aspects of the present invention, the antibody-like molecule comprises a CH3 domain of one heavy chain having the amino acid substitution Y349C / T366S / L368A / Y407 according to the EU amino acid numbering scheme for antibodies, and a CH3 domain of the other heavy chain having the amino acid substitution S354C / T366W according to the EU amino acid numbering scheme for antibodies.

[0065] In some aspects of the present invention, the antibody-like molecule includes an Fc fragment belonging to IgG. In some aspects of the present invention, the antibody-like molecule comprises an Fc fragment selected from the group including human IgG1, IgG2, or IgG4.

[0066] In some aspects of the present invention, the antibody-like molecule comprises an Fc fragment monomer containing substitutions that do not result in the characteristics of ADCC, CDC, and / or ADCP in the antibody-like molecule. In some aspects of the present invention, the antibody-like molecule comprises an Fc fragment monomer containing L234A and L235A substitutions according to the EU numbering scheme for antibody amino acids.

[0067] In some aspects of the present invention, the antibody-like molecule comprises an Fc fragment monomer containing a substitution that gives the antibody-like molecule a sustained effect. In some aspects of the present invention, the antibody-like molecule comprises an Fc fragment monomer containing substitutions M252Y, S254T, and T256E according to the EU numbering scheme for antibody amino acids.

[0068] In some aspects of the present invention, the antibody-like molecule comprises an Fc fragment monomer containing substitutions that result in enhanced ADCC, CDC, and / or ADCP properties in the antibody-like molecule. In some aspects of the present invention, the antibody-like molecule comprises an Fc fragment monomer containing an E345R substitution according to the EU numbering scheme for antibody amino acids.

[0069] The above mutations in the Fc fragment are numbered according to the EU numbering scheme for amino acids in antibody chains (Edelman, GM, et al., Proc. Natl. Acad. Sci. USA 63 (1969), pp. 78-85; Kabat, EA, et al., Sequences of Proteins of Immunological Interests, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, (1991)).

[0070] The term "effector function" of an antibody or antibody-like molecule refers to the biological activity attributable to the Fc region of the antibody or antibody-like molecule (either the native Fc region sequence or Fc region amino acid variants), which varies with the isotype of the antibody or antibody-like molecule. Examples of effector functions of antibodies or antibody-like molecules include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors, BCRs); and B cell activation.

[0071] "Antibody-dependent cytotoxicity" or "ADCC" refers to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies or antibody-like molecules on target cells, which then cause lysis or phagocytosis of those target cells.

[0072] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, these cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; in this case, PBMC cells and NK cells are preferred.

[0073] "Complement-dependent cytotoxicity" and "CDC" refer to the ability of a molecule to lyse its target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule that has formed a complex with a congener antigen.

[0074] In some aspects of the present invention, the antibody-like molecule specifically binds to first and second targets, where the target refers to a single antigen. In some aspects of the present invention, the antibody-like molecule specifically binds to first and second targets, where the targets are understood to mean two, three, or more structurally similar antigens.

[0075] Those skilled in the art will understand that antibodies and antibody-like molecules may cross-react due to the similarity of their antigen / target protein structures. For example, an antibody against programmed cell death protein 1 (PD-1) may cross-react with other members of the CD28 receptor family, including CD28, CTLA-4, ICOS, and BTLA. Similarly, an antibody against GITR (glucocorticoid-inducible TNFR-related protein) may cross-react with other representatives of the TNFRSF superfamily, including CD137, OX40, and CD27. Cross-reaction of therapeutic antibodies is usually avoided; for example, PCT application publication WO2006 / 121168 provides an antibody that binds to PD-1 but substantially does not bind to CD28, CTLA-4, and human ICOS. However, cross-reactivity is an important characteristic for some therapeutic antibodies; for example, the prior art provides ustekinumab, a monoclonal antibody that binds to the p40 subunit common to both IL12 and IL23 (Juliane Weber et al., Ustekinumab, BioDrugs, 2009, 23(1):53~61. doi:10.2165 / 00063030-200923010-00006). Antibodies containing two antigen-binding fragments are also known, in which case one of the antigen-binding fragments binds to two antigens, IL17A or IL17F, because these interleukins have a high degree of structural identity; therefore, these antibodies with two antigen-binding fragments are simultaneously specific to three antigens. For example, patent US10562967 provides an antibody comprising two antigen-binding fragments that is specific to three antigens, IL-23p19, IL-17A, and IL-17F, while PCT application publication WO2017188850 provides an antibody comprising two antigen-binding fragments that is specific to three antigens, TNF-alpha, IL-17A, and IL-17F.Based on the above, the target to which an antibody-like molecule according to the present invention binds is understood to mean one antigen (of protein or other properties) containing one epitope recognized and bound by the antigen-binding site of the antibody-like molecule, or multiple antigens (of protein or other properties) containing multiple epitopes recognized and bound by the antigen-binding site of the antibody-like molecule. If an antibody-like molecule contains multiple antigen-binding sites that recognize and bind to distinct targets (including cases where a single antigen-binding site can recognize and bind to multiple antigens), then such an antibody or antibody-like molecule is said to be multispecific (bispecific in the case of two antigens, triplicate in the case of three antigens, quadruplespecific in the case of four antigens, etc.).

[0076] An antibody-like molecule according to the present invention, comprising two antigen-binding fragments and specifically binding to at least three antigens, such as 3, 4, 5, 6, 7, 8, or 9 antigens, falls within the scope of the present invention.

[0077] In some aspects of the present invention, an antibody-like molecule specifically binds to first and second targets, where the first and second targets can be independently selected from the group including CD20, BCMA, PD-1, PD-L1, CD47, GD2, AXL, TGF beta, CSF1R, blood coagulation factor 9 (FIX), blood coagulation factor 10 (FX), TNF alpha, IL17A, IL17F, or CD3.

[0078] In some aspects of the present invention, the antibody-like molecule is a multispecific antibody-like molecule. In some aspects of the present invention, the antibody-like molecule is a bispecific, triplicate, quadruple, quintuple, hexaple, heptapoleque, octaple, or nupleple specific antibody-like molecule.

[0079] In some embodiments, the antibody-like molecule is a bivalent, bispecific antibody-like molecule. The materials of this application provide the following antibody-like molecules: 08-001, 08-002, 08-003, 08-004, 08-005, or 08-006.

[0080] 08-001 is an antibody-like molecule that specifically binds to PD1 and CD20, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to PD1 and includes the following: a) Light chain of the first antigen-binding fragment, where the light chain of the first antigen-binding fragment comprises the light chain variable domain (prolgolimab_VL) and the light chain constant domain of the antibody prolgolimab having Sequence ID No. 5; And, b) The heavy chain of the first antigen-binding fragment, wherein the heavy chain of the first antigen-binding fragment comprises the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 4 and the heavy chain constant domain of the antibody, and the heavy chain constant domain comprises the first (CH1) heavy chain constant domain and an Fc fragment monomer, wherein the Fc fragment monomer comprises the second (CH2) and third (CH3) heavy chain constant domains; and, 2) The second antigen-binding fragment specifically binds to CD20 and includes the following: a) The light chain of the second antigen-binding fragment, where the light chain of the second antigen-binding fragment has the variable light chain domain of the antibody ocrelizumab (ocrelizumab_VL) having SEQ ID NO: 7, It includes the constant domain, which is the α3 membrane proximal domain of the human CD1b protein, having the amino acid sequence of Sequence ID No. 2; And, b) The heavy chain of the second antigen-binding fragment, where the heavy chain of the second antigen-binding fragment is The heavy chain variable domain of the antibody ocrelizumab (ocrelizumab_VH), which has sequence number 6, The constant domain of the human CD1b protein, which is β2 microglobulin (β2M), has the amino acid sequence of Sequence ID No. 1, It comprises an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; It is an antibody-like molecule.

[0081] 08-001 is an antibody-like molecule that specifically binds to PD1 and CD20, and the antibody-like molecule is; 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to PD1 and includes the following: a) The light chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 11 (prorugolimab_VL_CK); And, b) The heavy chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 10 (prorugolimab_VH_HC_hole); and, 2) The second antigen-binding fragment specifically binds to CD20 and includes the following: a) The light chain of the second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 13 (ocrelizumab_VL_CD1b); and b) The heavy chain of the second antigen-binding fragment having the amino acid sequence of Sequence ID No. 12 (ocrelizumab_VH_b2m_Fc_knob); It is an antibody-like molecule.

[0082] 08-002 is an antibody-like molecule that specifically binds to CD20 and PD1, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to CD20 and includes the following: a) The light chain of the first antigen-binding fragment, wherein the light chain of the first antigen-binding fragment comprises the variable light chain domain of the antibody ocrelizumab (ocrelizumab_VL) having Sequence ID No. 7, and the constant light chain domain; And, b) The heavy chain of the first antigen-binding fragment, wherein the heavy chain of the first antigen-binding fragment comprises the heavy chain variable domain of the antibody ocrelizumab (ocrelizumab_VH) having SEQ ID NO: 6 and the heavy chain constant domain of the antibody, and the heavy chain constant domain comprises the first (CH1) heavy chain constant domain and an Fc fragment monomer, wherein the Fc fragment monomer comprises the second (CH2) and third (CH3) heavy chain constant domains; and, 2) The second antigen-binding fragment specifically binds to PD1 and includes the following: a) The light chain of the second antigen-binding fragment, where the light chain of the second antigen-binding fragment has the variable light chain domain of the antibody prorugolimab (prorugolimab_VL) having SEQ ID NO: 5, It includes the constant domain, which is the α3 membrane proximal domain of the human CD1b protein, having the amino acid sequence of Sequence ID No. 2; And, b) The heavy chain of the second antigen-binding fragment, where the heavy chain of the second antigen-binding fragment has the variable heavy chain domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 4, It comprises a constant domain, which is the β2 microglobulin (β2M) of the human CD1b protein having the amino acid sequence of Sequence ID No. 1, and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; It is an antibody-like molecule.

[0083] 08-002 is an antibody-like molecule that specifically binds to CD20 and PD1, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to CD20 and includes the following: a) The light chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 15 (ocrelizumab_VL_CK); And, b) The heavy chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 14 (ocrelizumab_VH_HC_hole); and, 2) The second antigen-binding fragment specifically binds to PD1 and includes the following: a) The light chain of the second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 17 (prorugolimab_VL_CD1b); And, b) The heavy chain of the second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 16 (prorugolimab_VH_b2m_Fc_knob); It is an antibody-like molecule.

[0084] 08-003 is an antibody-like molecule that specifically binds to PD1 and CSF1R, and the antibody-like molecule is; 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to PD1 and includes the following: a) Light chain of the first antigen-binding fragment, wherein the light chain of the first antigen-binding fragment comprises the light chain variable domain (progolimab_VL) and the light chain constant domain of the antibody prorugolimab having Sequence ID No. 5; And, b) The heavy chain of the first antigen-binding fragment, wherein the heavy chain of the first antigen-binding fragment comprises the heavy chain variable domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 4 and the heavy chain constant domain of the antibody, and the heavy chain constant domain comprises the first (CH1) heavy chain constant domain and an Fc fragment monomer, wherein the Fc fragment monomer comprises the second (CH2) and third (CH3) heavy chain constant domains; and, 2) The second antigen-binding fragment specifically binds to CSF1R and includes the following: a) The light chain of the second antigen-binding fragment, where the light chain of the second antigen-binding fragment has the light chain variable domain of the antibody against CSF1R (anti-CSF1R_VL) having Sequence ID No. 9, It includes the constant domain, which is the α3 membrane proximal domain of the human CD1b protein, having the amino acid sequence of Sequence ID No. 2; And, b) The heavy chain of the second antigen-binding fragment, where the heavy chain of the second antigen-binding fragment is The heavy chain variable domain (anti-CSF1R_VH) of the antibody against CSF1R having sequence number 8, The constant domain of the human CD1b protein, which is β2 microglobulin (β2M), has the amino acid sequence of Sequence ID No. 1, It comprises an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; It is an antibody-like molecule.

[0085] 08-003 is an antibody-like molecule that specifically binds to PD1 and CSF1R, and the antibody-like molecule is; 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to PD1 and includes the following: a) The light chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 11 (prorugolimab_VL_CK); And, b) The heavy chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 10 (prorugolimab_VH_HC_hole); and, 2) The second antigen-binding fragment specifically binds to CSF1R and includes the following: a) The light chain of the second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 19 (anti-CSF1R_VL_CD1b); And, b) The heavy chain of the second antigen-binding fragment having the amino acid sequence of Sequence ID No. 18 (anti-CSF1R_VH_b2m_Fc_knob); It is an antibody-like molecule.

[0086] 08-004 is an antibody-like molecule that specifically binds to CSF1R and PD1, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to CSF1R and includes the following: a) The light chain of the first antigen-binding fragment, where the light chain of the first antigen-binding fragment has the light chain variable domain of the antibody against CSF1R (anti-CSF1R_VL) having SEQ ID NO: 9, Includes a light chain constant domain; And, b) The heavy chain of the first antigen-binding fragment, where the heavy chain of the first antigen-binding fragment is The antibody having sequence number 8 comprises a heavy chain variable domain (anti-CSF1R_VH) and a heavy chain constant domain of the antibody against CSF1R, wherein the heavy chain constant domain comprises a first (CH1) heavy chain constant domain and an Fc fragment monomer, the Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; and, 2) The second antigen-binding fragment specifically binds to PD1 and includes the following: a) The light chain of the second antigen-binding fragment, where the light chain of the second antigen-binding fragment has the variable light chain domain of the antibody prorugolimab (prorugolimab_VL) having SEQ ID NO: 5, It includes the constant domain, which is the α3 membrane proximal domain of the human CD1b protein, having the amino acid sequence of Sequence ID No. 2; And, b) The heavy chain of the second antigen-binding fragment, where the heavy chain of the second antigen-binding fragment has the variable heavy chain domain of the antibody prorugolimab (prorugolimab_VH) having SEQ ID NO: 4, The constant domain of the human CD1b protein, which is β2 microglobulin (β2M), has the amino acid sequence of Sequence ID No. 1, It comprises an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; It is an antibody-like molecule.

[0087] 08-004 is an antibody-like molecule that specifically binds to CSF1R and PD1, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to CSF1R and includes the following: a) The light chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 21 (anti-CSF1R_VL_CK); And, b) The heavy chain of the first antigen-binding fragment having the amino acid sequence of Sequence ID No. 20 (anti-CSF1R_VH_HC_hole); and, 2) The second antigen-binding fragment specifically binds to PD1 and includes the following: a) The light chain of the second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 17 (prorugolimab_VL_CD1b); And, b) The heavy chain of the second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 16 (prorugolimab_VH_b2m_Fc_knob); It is an antibody-like molecule.

[0088] 08-005 is an antibody-like molecule that specifically binds to CD20 and CSF1R, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to CD20 and includes the following: a) The light chain of the first antigen-binding fragment, where the light chain of the first antigen-binding fragment has the variable light chain domain of the antibody ocrelizumab (ocrelizumab_VL) having SEQ ID NO: 7, Includes a light chain constant domain; And, b) The heavy chain of the first antigen-binding fragment, wherein the heavy chain of the first antigen-binding fragment comprises the heavy chain variable domain of the antibody ocrelizumab (ocrelizumab_VH) having SEQ ID NO: 6 and the heavy chain constant domain of the antibody, and the heavy chain constant domain comprises the first (CH1) heavy chain constant domain and an Fc fragment monomer, wherein the Fc fragment monomer comprises the second (CH2) and third (CH3) heavy chain constant domains; and, 2) The second antigen-binding fragment specifically binds to CSF1R and includes the following: a) The light chain of the second antigen-binding fragment, where the light chain of the second antigen-binding fragment has the light chain variable domain of the antibody against CSF1R (anti-CSF1R_VL) having Sequence ID No. 9, It includes the constant domain, which is the α3 membrane proximal domain of the human CD1b protein, having the amino acid sequence of Sequence ID No. 2; And, b) The heavy chain of the second antigen-binding fragment, where the heavy chain of the second antigen-binding fragment is The heavy chain variable domain (anti-CSF1R_VH) of the antibody against CSF1R having sequence number 8, The constant domain of the human CD1b protein, which is β2 microglobulin (β2M), has the amino acid sequence of Sequence ID No. 1, It comprises an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; It is an antibody-like molecule.

[0089] 08-005 is an antibody-like molecule that specifically binds to CD20 and CSF1R, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to CD20 and includes the following: a) The light chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 15 (ocrelizumab_VL_CK); And, b) The heavy chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 14 (ocrelizumab_VH_HC_hole); and, 2) The second antigen-binding fragment specifically binds to CSF1R and includes the following: a) The light chain of the second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 19 (anti-CSF1R_VL_CD1b); And, b) The heavy chain of the second antigen-binding fragment having the amino acid sequence of Sequence ID No. 18 (anti-CSF1R_VH_b2m_Fc_knob); It is an antibody-like molecule.

[0090] 08-006 is an antibody-like molecule that specifically binds to CSF1R and CD20, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to CSF1R and includes the following: a) The light chain of the first antigen-binding fragment, where the light chain of the first antigen-binding fragment has the light chain variable domain of the antibody against CSF1R (anti-CSF1R_VL) having SEQ ID NO: 9, Includes a light chain constant domain; And, b) The heavy chain of the first antigen-binding fragment, where the heavy chain of the first antigen-binding fragment is The antibody having sequence number 8 comprises a heavy chain variable domain (anti-CSF1R_VH) and a heavy chain constant domain of the antibody against CSF1R, wherein the heavy chain constant domain comprises a first (CH1) heavy chain constant domain and an Fc fragment monomer, the Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; and, 2) The second antigen-binding fragment specifically binds to CD20 and includes the following: a) The light chain of the second antigen-binding fragment, where the light chain of the second antigen-binding fragment has the variable light chain domain of the antibody ocrelizumab (ocrelizumab_VL) having SEQ ID NO: 7, It includes the constant domain, which is the α3 membrane proximal domain of the human CD1b protein, having the amino acid sequence of Sequence ID No. 2; And, b) The heavy chain of the second antigen-binding fragment, where the heavy chain of the second antigen-binding fragment is The heavy chain variable domain of the antibody ocrelizumab (ocrelizumab_VH), which has sequence number 6, The constant domain of the human CD1b protein, which is β2 microglobulin (β2M), has the amino acid sequence of Sequence ID No. 1, It comprises an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains; It is an antibody-like molecule.

[0091] 08-006 is an antibody-like molecule that specifically binds to CSF1R and CD20, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment and 2) a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to CSF1R and includes the following: a) Light chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 21 (anti-CSF1R_VL_CK); And, b) The heavy chain of the first antigen-binding fragment having the amino acid sequence of Sequence ID No. 20 (anti-CSF1R_VH_HC_hole); and, 2) The second antigen-binding fragment specifically binds to CD20 and includes the following: a) The light chain of the second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 13 (ocrelizumab_VL_CD1b); And, b) Containing a heavy chain of a second antigen-binding fragment (ocrelizumab_VH_b2m_Fc_knob) having the amino acid sequence of SEQ ID NO: 12; It is an antibody-like molecule.

[0092] Such antibody-like molecules are provided for illustrative purposes to demonstrate the operability of the format of antibody-like molecules according to the present invention, as well as their remarkable properties. Such antibody-like molecules should not be construed as limiting the antibody-like molecules of the present invention in any way.

[0093] The yield parameters of products having precise heterodimer assembly of two distinct heavy chains and precise pairing between two distinct light chains and cognate heavy chains are independent of the heavy and light chain variable fragments of the antibody-like molecule and its specificity to the antigen.

[0094] Antibody-like molecules according to the present invention can be used to treat various diseases, particularly neoplastic diseases, autoimmune diseases, or diseases associated with blood coagulation (clotting) disorders. nucleic acid molecule In one aspect, the present invention relates to nucleic acids encoding the above-mentioned antibody-like molecules.

[0095] The terms “nucleic acid,” “nucleic sequence,” “nucleic acid sequence,” “polynucleotide,” “oligonucleotide,” “polynucleotide sequence,” and “nucleotide sequence” are used interchangeably herein, but mean the exact sequence of modified or unmodified nucleotides that determines a fragment or region of nucleic acid, which may or may not contain non-natural nucleotides, and which may be double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcript of said DNA.

[0096] Unless otherwise specified, the term nucleotide sequence includes its complement. Therefore, a nucleic acid having a particular sequence should be understood to include its complementary strand having that complementary sequence.

[0097] In any of the above embodiments, nucleic acid molecules may be isolated. An "isolated" nucleic acid molecule is one that is identified and separated from at least one other nucleic acid molecule—an impurity. An isolated nucleic acid molecule differs from the form or set it would appear in under natural conditions. Therefore, an isolated nucleic acid molecule is different from the nucleic acid molecules present in cells under natural conditions.

[0098] In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 22-33. The nucleic acid molecule may also contain any combination of the nucleotide sequences.

[0099] In some aspects of the present invention, the isolated nucleic acid is DNA. In one embodiment, the present invention relates to a nucleic acid molecule encoding an amino acid sequence of the light chain or heavy chain of the antibody-like molecule according to the present invention, selected from the following: - A nucleic acid comprising a light chain of a first antigen-binding fragment that comprises a light chain variable domain and a light chain constant domain, and encoding the amino acid sequence of the light chain; - The heavy chain of the first antigen-binding fragment comprises the antibody heavy chain variable domain and the antibody heavy chain constant domain comprising an Fc fragment monomer containing the first (CH1) heavy chain constant domain and the second (CH2) and third (CH3) heavy chain constant domains. A nucleic acid encoding the amino acid sequence of the heavy chain described above; - The light chain of the second antigen-binding fragment consists of a light chain variable domain and The constant domain is β2 microglobulin (β2M) of human CD1b (differentiation antigen group 1) protein, having the amino acid sequence of Sequence ID No. 1, Nucleic acid encoding the amino acid sequence of the above light chain; - The heavy chain of the second antigen-binding fragment consists of a heavy chain variable domain and It comprises a constant domain, which is the α3 membrane proximal domain of the human CD1b protein having the amino acid sequence of SEQ ID NO: 2, and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains. A nucleic acid encoding the amino acid sequence of the heavy chain described above; - The light chain of the second antigen-binding fragment consists of a light chain variable domain and The constant domain, which is the α3 membrane proximal domain of the human CD1b protein, has the amino acid sequence of SEQ ID NO: 2, and Nucleic acid encoding the amino acid sequence of the above light chain; or - The heavy chain of the second antigen-binding fragment consists of a heavy chain variable domain and It comprises a constant domain, which is the β2 microglobulin (β2M) of the human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains. A nucleic acid that encodes the amino acid sequence of the heavy chain described above.

[0100] The nucleic acid molecule may also include the above-mentioned combination of nucleotide sequences necessary for producing an antibody-like molecule according to the present invention. As those skilled in the art will understand, due to the duplication of the genetic code, various different DNA sequences can encode the amino acid sequences of the light or heavy chains of antibody-like molecules according to the present invention, or fragments thereof (VH, VL, CDR, etc.). Creating these alternative DNA sequences that encode a single identical amino acid sequence is well within the scope of the art of those trained in the art. Such variant DNA sequences are within the scope of the present invention.

[0101] Nucleic acid molecules according to the present invention can be isolated from any source that produces antibody-like molecules according to the present invention. In certain embodiments of the present invention, nucleic acid molecules of the present invention can be synthesized by chemical synthesis rather than by isolation.

[0102] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain (prorugolimab_VH_HC_hole) of the first antigen-binding fragment of antibody-like molecules 08-001 and 08-003 and includes the nucleotide sequence of SEQ ID NO: 22.

[0103] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain (prorugolimab_VL_CK) of the first antigen-binding fragment of antibody-like molecules 08-001 and 08-003 and includes the nucleotide sequence of SEQ ID NO: 23.

[0104] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain (ocrelizumab_VH_b2m_Fc_knob) of the second antigen-binding fragment of antibody-like molecules 08-001 and 08-006 and includes the nucleotide sequence of SEQ ID NO: 24.

[0105] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain (ocrelizumab_VL_CD1b) of the second antigen-binding fragment of antibody-like molecules 08-001 and 08-006 and includes the nucleotide sequence of SEQ ID NO: 25.

[0106] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain (ocrelizumab_VH_HC_hole) of the first antigen-binding fragment of antibody-like molecules 08-002 and 08-005 and includes the nucleotide sequence of SEQ ID NO: 26.

[0107] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain (ocrelizumab_VL_CK) of the first antigen-binding fragment of antibody-like molecules 08-002 and 08-005 and includes the nucleotide sequence of SEQ ID NO: 27.

[0108] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain (prorugolimab_VH_b2m_Fc_knob) of the second antigen-binding fragment of antibody-like molecules 08-002 and 08-004 and includes the nucleotide sequence of SEQ ID NO: 28.

[0109] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain (prorugolimab_VL_CD1b) of the second antigen-binding fragment of antibody-like molecules 08-002 and 08-004 and includes the nucleotide sequence of SEQ ID NO: 29.

[0110] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain (anti-CSF1R_VH_b2m_Fc_knob) of the second antigen-binding fragment of antibody-like molecules 08-003 and 08-005 and includes the nucleotide sequence of SEQ ID NO: 30.

[0111] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain (anti-CSF1R_VL_CD1b) of the second antigen-binding fragment of antibody-like molecules 08-003 and 08-005 and includes the nucleotide sequence of SEQ ID NO: 31.

[0112] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the heavy chain (anti-CSF1R_VH_HC_hole) of the first antigen-binding fragment of antibody-like molecules 08-004 and 08-006 and includes the nucleotide sequence of SEQ ID NO: 32.

[0113] In some aspects of the present invention, the nucleic acid is a nucleic acid that encodes the amino acid sequence of the light chain (anti-CSF1R_VL_CK) of the first antigen-binding fragment of antibody-like molecules 08-004 and 08-006 and comprises the nucleotide sequence of SEQ ID NO: 33.

[0114] Nucleic acid molecules can be used to express antibody-like molecules according to the present invention. vector In one aspect, the present invention relates to an expression vector comprising any one of the nucleic acid molecules that encode the corresponding amino acid sequence of an antibody-like molecule according to the present invention. The present invention relates to a vector suitable for the expression of any one of the nucleotide sequences described herein.

[0115] As used herein, the term "vector" means a nucleic acid molecule that can be linked to another nucleic acid to transport it. As used herein, the term “expression” is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0116] The present invention relates to a vector comprising a nucleic acid molecule that encodes any one of the above antibody-like molecules or a part thereof, selected from the following: - A nucleic acid comprising a light chain of a first antigen-binding fragment that comprises a light chain variable domain and a light chain constant domain, and encoding the amino acid sequence of the light chain; - The heavy chain of the first antigen-binding fragment comprises the antibody heavy chain variable domain and the antibody heavy chain constant domain comprising an Fc fragment monomer containing the first (CH1) heavy chain constant domain and the second (CH2) and third (CH3) heavy chain constant domains. A nucleic acid encoding the amino acid sequence of the heavy chain described above; - The light chain of the second antigen-binding fragment consists of a light chain variable domain and The constant domain is β2 microglobulin (β2M) of human CD1b (differentiation antigen group 1) protein, having the amino acid sequence of Sequence ID No. 1, Nucleic acid encoding the amino acid sequence of the above light chain; - The heavy chain of the second antigen-binding fragment consists of a heavy chain variable domain and It comprises a constant domain, which is the α3 membrane proximal domain of the human CD1b protein having the amino acid sequence of SEQ ID NO: 2, and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains. A nucleic acid encoding the amino acid sequence of the heavy chain described above; - The light chain of the second antigen-binding fragment consists of a light chain variable domain and The constant domain, which is the α3 membrane proximal domain of the human CD1b protein, has the amino acid sequence of SEQ ID NO: 2, and Nucleic acid encoding the amino acid sequence of the above light chain; or - The heavy chain of the second antigen-binding fragment consists of a heavy chain variable domain and It comprises a constant domain, which is the β2 microglobulin (β2M) of the human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer containing the second (CH2) and third (CH3) heavy chain constant domains. A nucleic acid that encodes the amino acid sequence of the heavy chain described above.

[0117] In some aspects of the present invention, the vector is a plasmid, i.e., a circular double-stranded piece of DNA into which further DNA segments can be inserted. In some aspects of the present invention, the vector is a viral (expression) vector, in which case further DNA segments may be inserted into the viral genome.

[0118] In some aspects of the present invention, vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal vectors having a bacterial site as an origin of replication). In further aspects of the present invention, vectors (e.g., non-episomal vectors) may integrate into the host cell's genome upon introduction into the host cell, thereby replicating together with host genes. Furthermore, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).

[0119] In some aspects of the present invention, the expression vector may include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, cosmids, YACs, etc. DNA molecules may be inserted into the vector so that the transcriptional and translational regulatory sequences within the vector perform their intended functions of regulating DNA transcription and translation. The expression vector and expression regulatory sequences may be selected to suit the expression host cell in which they are used.

[0120] In some aspects of the present invention, DNA molecules that partially or completely encode the heavy chain and light chain sequences can be inserted into separate vectors. In one embodiment, any combination of the above DNA molecules is introduced into the same expression vector.

[0121] In one aspect of the present invention, a DNA molecule can be introduced into an expression vector by a standard method (for example, ligation of a gene fragment of an antibody or antibody-like molecule and a complementary restriction site on the vector, or, if no restriction site exists, blunt-end ligation).

[0122] In some aspects of the present invention, a suitable vector is one that includes a restriction site into which any VH or VL sequence can be readily inserted and expressed, as described above. The recombinant expression vector may also encode a signal peptide that facilitates the secretion of an antibody-like molecular chain from a host cell. The gene for the antibody-like molecular chain can be cloned into the vector such that the signal peptide is in-frame linked to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0123] In some aspects of the present invention, the vector may contain expression regulatory sequences. As used herein, the term “expression regulatory sequence” refers to a polynucleotide sequence necessary for the expression and processing of the coding sequence into which they are inserted. Those skilled in the art will understand that the design of an expression vector, including the selection of expression regulatory sequences, may depend on factors such as the selection of the type of host cell to be transformed, the required expression level of the antibody or antibody-like molecule, and so on. Expression regulatory sequences include appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if necessary, sequences that enhance protein secretion. The nature of such expression regulatory sequences varies depending on the host organism; in prokaryotes, such expression regulatory sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such expression regulatory sequences typically include promoters and transcription termination sequences. Preferred expression regulatory sequences for mammalian host cells include viral elements that ensure high levels of protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), Simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), promoters and / or enhancers derived from polyomaviruses, as well as potent mammalian promoters such as TTR promoters, native immunoglobulin promoters, or actin promoters. Expression regulatory sequences encompass at least all components whose presence is important for expression and processing.

[0124] In some aspects of the present invention, in addition to the gene and expression regulatory sequence of the antibody-like molecular chain, the recombinant expression vector of the present invention may carry further sequences, such as sequences that regulate vector replication in host cells (e.g., origin of replication) and selection marker genes. The selection marker genes facilitate the selection of host cells into which the vector has been introduced.

[0125] host cell In one aspect, the present invention relates to a method for producing a host cell that produces any one of the antibody-like molecules according to the present invention, comprising the step of transforming the cell with one or more vectors containing the combination of nucleotide sequences necessary for producing the antibody-like molecule according to the present invention.

[0126] In one aspect, the present invention relates to a host cell that produces any one of the antibody-like molecules according to the present invention, comprising the combination of nucleotide sequences necessary for producing the antibody-like molecule according to the present invention.

[0127] As used herein, the term “host cell” refers to a cell into which a recombinant expression vector has been introduced. The present invention relates to a host cell that may, for example, contain the above vector according to the present invention. The present invention further relates to a host cell that includes, for example, a nucleotide sequence encoding a heavy chain, a nucleotide sequence encoding a light chain, or both. It should be understood that “host cell” refers not only to a particular cell of interest but also to the offspring of such a cell. Since modifications may occur in later generations due to either mutation or environmental influences, such offspring may, in fact, not be identical to the parent cell; however, such cells still fall within the scope of the term “host cell” as used herein.

[0128] Nucleic acid molecules encoding the antibody-like molecules described above according to the present invention, and vectors containing these nucleic acid molecules, can be used for the transformation of mammalian cells, plant cells, bacterial cells, or yeast cells. Transformation can be carried out by any known technique for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybren-mediated transfection, protoplast fusion, liposome-mediated polynucleotide encapsulation, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral (expression) vectors.

[0129] Mammalian cell lines used as hosts for transformation are well known in the art and include several immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, SK-HEP1 cells, HUH7 cells, Hep-RG cells, and several other cell lines. Cell lines are selected by determining which cell lines have high expression levels and provide the required characteristics of the proteins they produce. Other cell lines that may be used are insect cell lines such as Sf9 cells or Sf21 cells. When a recombinant expression vector encoding the antibody-like molecule of the present invention is introduced into a mammalian host cell, the antibody-like molecule is produced by means of culturing the host cell for a period of time sufficient to express the antibody-like molecule of the present invention in the host cell, or more preferably, to secrete the antibody-like molecule into the culture medium in which the host cell is cultured. The antibody-like molecule of the present invention can be isolated from the culture medium using standard protein purification techniques. Plant host cells include, for example, tobacco (Nicotiana), Arabidopsis, duckweed, maize, wheat, potato, etc. Bacterial host cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0130] Furthermore, the production level of the antibody-like molecules of the present invention from the producing cell line can be enhanced using several known techniques. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions.

[0131] The antibody-like molecules of the present invention in different cell lines may have different glycosylation patterns. However, the antibody-like molecules of the present invention encoded by the nucleic acid molecules described herein, or comprising amino acid sequences provided herein, are part of the present invention regardless of the glycosylation of the binding molecule and, generally, regardless of the presence or absence of post-translational modifications.

[0132] The host cells mentioned above are unrelated to host cells created using human embryos. The host cells mentioned above are unrelated to host cells created by modifying the genetic integrity of human germline cells.

[0133] Method for producing antibody-like molecules In one aspect, the present invention relates to a method for producing any one of the above-mentioned antibody-like molecules, wherein the method is: a) A step in which host cells are transformed. - Using an expression vector containing nucleic acid molecules encoding the light and heavy chains of the first antigen-binding fragment of an antibody-like molecule, - Using an expression vector containing nucleic acid molecules encoding the light and heavy chains of the second antigen-binding fragment of an antibody-like molecule, Transformation step, b) A step of culturing host cells under conditions suitable for the synthesis of the antibody-like molecule, and c) A step of isolating the antibody-like molecule from the cell culture, Includes. [Examples]

[0134] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0135] Although the aforementioned inventions have been described in some detail by examples and illustrations for clarity, it will be readily apparent to those skilled in the art, in light of the teachings of the present invention, that certain changes and modifications can be made to them without departing from the spirit or scope of the appended embodiments.

[0136] Materials and general methods Recombinant DNA techniques DNA was manipulated using standard methods, as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's protocols.

[0137] gene synthesis The desired gene segment was prepared from oligonucleotides synthesized by chemical synthesis. Gene segments of 300–4,000 bp in length, flanked by singular restriction sites, were assembled by oligonucleotide annealing and ligation, including PCR amplification, and then cloned through specific restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0138] DNA sequencing The DNA sequence was determined by Sanger sequencing. DNA and protein sequence analysis and sequence data management The Ylab2 (Biocad) software package was used for sequence creation, mapping, analysis, annotation, and illustration.

[0139] Expression vector Expression plasmid variants were applied to transient expression of the antibody, antibody-like protein, and antigen of the present invention in eukaryotic cells (e.g., CHO cells). In addition to the expression cassette for the target protein, the (plasmid) vector contained: a replication origin enabling replication of the plasmid in Escherichia coli (E. coli), and genes conferring resistance to various antibiotics (e.g., ampicillin and kanamycin) in E. coli.

[0140] Fusion genes containing the described strands of the antibody or antibody-like molecule were generated by PCR and / or gene synthesis and assembled by known methods and techniques, for example, using unique restriction sites in the corresponding vector, by means of connecting the corresponding nucleic acid segments. The subcloned nucleic acid sequences were validated by DNA sequencing. The required amount of plasmid for transient transfection was generated in E. coli cell cultures and isolated using known techniques.

[0141] Generation and purification of recombinant antigens in suspension cultures of mammalian cells Recombinant proteins were produced using cells from an established cell line derived from Chinese hamster ovary cells (CHO line). Suspension culture was performed in flasks on an orbital incubator shaker using serum-free medium supplemented with 8 mM L-glutamine and Pluronic® 68 1 g / l. For transient expression, concentrations of 2–2.2 × 10⁶ were observed. 6 Cells were transfected with linear polyethyleneimine at a concentration of 100 cells / ml. Nine days after transfection, the culture medium was separated from the cells by filtration through a 0.22 μm filter.

[0142] Histidine-tagged proteins were purified by metal chelate chromatography. The purified proteins were filtered through a 0.22 μm filter and stored at -70°C. The purity of the resulting protein solution was evaluated using SDS gel electrophoresis. Electrophoresis was performed in denatured 12% PAAG in the presence of mercaptoethanol and in denatured 7.5% PAAG in the absence of mercaptoethanol.

[0143] Production of antibodies and antibody-like proteins in suspension culture of mammalian cells Control antibodies and antibody-like molecules according to the present invention were prepared using cells from established cell lines obtained from Chinese hamster ovary cells (CHO lineage). Suspension culture was performed in flasks on an orbital incubator shaker using serum-free medium supplemented with 8 mM L-glutamine and Pluronic® 68 1 g / l. In the case of transient expression, concentrations of 2-2.2 × 10⁶ were observed. 6 Cells were transfected using linear polyethyleneimine (PEI) at a concentration of 10 cells / ml. The DNA / PEI ratio was 1:3 / 1:10. Nine days after transfection, the culture medium was separated from the cells by filtration through a 0.5 / 0.22 μm deep-bed filter, and protein titer was then measured using a ForteBio with a standard method. The clean culture medium was passed through a protein A affinity adsorbent column with 10-20 mg of adsorbent per ml (the column was equilibrated with phosphate-buffered saline (PBS, pH 7.4)). The column was then washed with 5 times the column volume of PBS to remove nonspecifically bound components. The bound proteins were eluted using 0.1 M glycine buffer (pH 3). The major protein elution peak was collected and adjusted to pH 6.0 with 1 M Tris buffer (pH 8). All steps were performed at a flow rate of 110 cm / h. Next, the protein was dialyzed into acetate buffer (pH 5.0), filtered (0.22 μm), transferred to a tube, and stored at -70°C.

[0144] The purity of the resulting protein solution was evaluated using SDS gel electrophoresis under reducing and non-reducing conditions, as well as using size exclusion high-performance liquid chromatography (SE HPLC).

[0145] SE HPLC was performed using a TSK-Gel G3000SWXL column, 7.8 × 300 mm, particle size: 5 μm, pore size: 250 Å, and a TSK-Gel Guard SWxl pre-column.

[0146] Example 1. Selection of CH1 / CK domain substitution in antibody-like molecules To ensure proper heterodimerization of the heavy and light chains within the antibody-like molecule, one of the two pairs of CH1 / CK domains was replaced with a structurally identical domain derived from another protein.

[0147] This resulted in a dimerization unit consisting of 1) the native sequence of human CD1b protein β2 microglobulin (β2M) having the amino acid sequence of SEQ ID NO: 1, and 2) the α3 membrane proximal domain of human CD1b having the S12K and N59D mutations and GSC elongation at the C-terminus, having the amino acid sequence of SEQ ID NO: 2. Such a dimerization unit within the antibody-like molecule made it possible to produce functional antibody-like molecules of sufficient purity with separate variable domains (shown in the examples below), and also improved the temperature stability of the above molecules and the yield of accurately assembled antibody-like molecules.

[0148] Example 2. Preparation of gene constructs for antibody-like molecule generation. To construct constructs encoding the light and heavy chain sequences of the first antigen-binding fragment of an antibody-like molecule that specifically binds to the first target, PCR products containing the variable domain genes of the heavy and light chains of the antibody-like molecule were generated using primers containing restriction sites. The heavy chain variable domain was cloned into the vector pSXn-HChole-NR_VH1 using the SalI / XbaI restriction site. The light chain variable domain was cloned into the vector pSXn-CL-BR_VLl using the SalI / XbaI restriction site.

[0149] To construct a structure encoding the light and heavy chain sequences of a second antigen-binding fragment of an antibody-like molecule that specifically binds to a second target, a construct containing sequences encoding the variable domains of the heavy and light chains of the antibody-like molecule was synthesized and fused to the membrane-proximal domain of the human CD1b protein, including modifications (https: / / www.rcsb.org / structure / 5wl1).

[0150] The above sequences were synthesized from oligonucleotides by PCR using primers containing restriction sites. A heavy chain variable domain containing the first membrane proximal domain of the human CD1b sequence, including modifications, was cloned into the vector pSX-FCknob-PR using the SalI / XbaI restriction site. A light chain variable domain containing the second membrane proximal domain of the human CD1b sequence, with or without modifications, was cloned into the vector pSX-HR using the SalI / XbaI restriction site.

[0151] The four types of vectors described above were combined during the transfection stage to produce antibody-like molecules according to the present invention. All required quantities of the above plasmid were generated in E. coli cells and purified using the Maxiprep Qiagen kit.

[0152] Example 3. Generation of an antibody-like molecule containing a human CD1b protein dimerization unit. To check the universality of the present invention's approach as a platform solution for assembling antibody-like molecules using any pair of antigen-binding fragments (hereinafter referred to as variable light and heavy chain fragments), the inventors selected three random pairs of variable light and heavy chain fragments from known antibodies (see Table 1) and used them to generate six antibody-like molecules. Table 1 shows the results of the productivity of the generated proteins. Figures 1 and 2 show the results of SDS gel electrophoresis of the samples generated under non-reducing and reducing conditions. For all six samples under non-reducing conditions, a major band is present at approximately 150 kDa, corresponding to the full-length molecule. Table 1 also shows the results of the sample purity by SE HPLC.

[0153] [Table 1]

[0154] Therefore, antibody-like molecules comprising a dimerization unit according to the present invention, having various combinations of antigen-binding fragments, exhibit high purity and high productivity. Example 4. Determination of affinity of full-length antibody-like molecules in Forte Bio Octet RED 384. To confirm that the generated antibody-like molecules had not lost their antigen-binding ability, the inventors performed affinity analysis using Forte Bio Octet RED 384. For antibody-like molecules 08-001 and 08-002, the inventors measured their affinity for the extracellular domain of human PD-1 protein (hPD1ex-H6F) and their affinity for the biotinylated peptide [NH2]CEPANPSEKNSPSTQYCYSIQS[CH2CH2]biotin (hereinafter referred to as CD20 peptide), which contains a fragment of the human CD20 sequence in its amino acid sequence; for antibody-like molecules 08-003 and 08-004, the inventors measured their affinity for the extracellular domain of human PD-1 protein (hPD1ex-H6F) and their affinity for the extracellular domain of human CSF1R protein (hCSF1R_His); and for antibody-like molecules 08-005 and 08-006, the inventors measured their affinity for the extracellular domain of human CSF1R protein (hCSF1R_His) and their affinity for the CD20 peptide.

[0155] As the hCSF1R antigen, the inventors used amino acid sequence 20-512 (hereinafter referred to as AA) of a human CSF1R protein (macrophage colony-stimulating factor 1 receptor (Homo sapiens), UNIPROT ID P07333) with a molecular weight of 57.4 kDa and a C-terminal His tag and FLAG tag. As the hPD-1ex-H6F antigen, the inventors used AA21-170 sequence of a human PD-1 protein (programmed cell death protein 1 (Homo sapiens), UNIPROT ID Q15116) with a molecular weight of 20.6 kDa and a C-terminal His tag and FLAG tag.

[0156] The gene sequence encoding the antigen was synthesized de novo, cloned into an expression vector, generated in CHO cells, and purified using affinity chromatography as described in the general example.

[0157] The experiment was conducted using a kinetic buffer solution (hereinafter referred to as 1×KB); the volume fraction of added Tween20 was 0.1%; the mass fraction of added BSA (bovine serum albumin) was 0.1%; pH 7.4. Before measurement, the ProA sensor was regenerated in a 50 mM glycine and hydrochloric acid solution at pH 1.8 (5 seconds in the regeneration solution, 5 seconds in 1×KB, repeated 3 times).

[0158] For the hPD1ex-H6F antigen and hCSF1R_His antigen, the protein A (ProA) biosensor (ForteBio) was immobilized by immersing it in a solution containing an antibody-like molecule at a concentration of 10 μg / ml for 300 seconds. The baseline was recorded at 1 × KB for 120 seconds. Next, the sensor loaded with the antibody-like molecule was immersed in a well containing a kinetic buffer solution of the target antigen (analyte) for 300 seconds. Measurements were performed at a concentration of 10 μg / ml (485.4 nM) for the hPD1ex-H6F analyte solution and at a concentration of 10 μg / ml (174.2 nM) for the hCSF1R_His analyte solution. Then, the dissociation of the complex at 1 × KB was detected for 600 seconds.

[0159] The reference sensor completed all stages except the binding stage, just as the sensor used to record the sensorgram of the analyte had completed. In the binding stage, the sensor was immersed in a 1 × KB solution that did not contain the analyte (the signal from the reference sensor was measured in parallel with the recording of the main sensorgram). The reference signal was subtracted from the signal received by the sensor that interacted with the analyte during the processing of the sensorgram.

[0160] To check for nonspecific interactions between the analyte and the sensor, the inventors used a sensor that was not loaded with an antibody-like molecule (in the loading stage, the sensor was immersed in a 1 × KB solution; all other steps were the same as those used for a sensor loaded with an antibody-like molecule).

[0161] For biotinylated CD20 peptide, a 2.5 μg / ml peptide was immobilized on the surface of a SAX sensor (High Precision Streptavidin (SAX) biosensor, ForteBio) for 300 seconds. The baseline was recorded in a 1 × KB solution for 120 seconds. The peptide-loaded sensor was then immersed in a well containing a 1 × KB solution of antibody-like molecules (analyte) for 300 seconds. Measurements were performed for solutions containing 300 μg / ml and 75 μg / ml of the analyte (antibody-like molecules in the case of CD20 peptide). The dissociation of the complex was recorded in a 1 × KB solution for 60 seconds.

[0162] All measurements were performed at 30°C, and the orbital mixing speed was set to 1000 revolutions per minute. To obtain numerical values ​​for the rate constants (kon is the on / binding rate constant, kdis is the dissociation rate constant, and KD is the equilibrium dissociation constant or affinity constant), the generated sensorgrams were processed according to a 1:1 interaction model using Global Fit in ForteBio Octet Data Analysis 9.0 software (a selection of one set of kon, kdis, and KD constants for analyzing several sensorgrams at different concentrations). The results are shown in Table 2.

[0163] [Table 2]

[0164] Table 3 shows the results of the verification regarding nonspecific interactions between the analyte and the non-loading sensor. No nonspecific interactions were detected between the analyte and the non-loading sensor.

[0165] [Table 3]

[0166] The results provided indicate that all antibody-like molecules 08-001 to 08-006 demonstrate specific binding to the target antigen; furthermore, the KD values ​​are of the same or similar magnitude regardless of the position of the corresponding antigen-binding fragment (as part of the Fab fragment, or as part of a Fab-like fragment containing the β2 microglobulin and modified α3 ​​domain of the CD1b protein instead of the CK and CH1 domains).

[0167] Example 5. Analysis of simultaneous binding of antibody-like molecules according to the present invention to two antigens. The simultaneous binding of antibody-like molecules 08-003 and 08-004 to two distinct target antigens (hPD1ex-H6F and hCSF1R_His) was analyzed on a Forte Bio Octet RED 384. The experiments were performed using an AR2G sensor (Amine Reactive Second-Generation (AR2G) biosensor, ForteBio). The experimental steps are shown in Table 4.

[0168] [Table 4]

[0169] The sensor was activated for 300 seconds with an aqueous solution containing 20 mM EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 10 mM sNHS (N-hydroxysulfosuccinimide). The antigen (hPD1ex-H6F) was loaded onto the surface of the biosensor for 300 seconds in 10 mM sodium acetate buffer, pH 5.0. The loading protein concentration was 10 μg / ml. The unreacted active site on the sensor surface was quenched for 300 seconds in a 1 M aqueous solution of ethanolamine, pH 8.5. The baseline for the fifth stage of the experiment and all subsequent stages were performed in kinetic buffer solution (1 × KB); the volume fraction of added Tween20 was 0.1%; the mass fraction of added BSA was 0.1%; pH 7.4. After recording the baseline (step 5), the antibody-like molecule was loaded onto the sensor; the concentration of the loaded antibody-like molecule was 10 μg / ml (step 6). A third baseline was then recorded (step 7). This step clearly shows no rapid signal decay, which indicates a specific interaction between the loaded antibody-like molecule and the hPD1ex-H6F previously immobilized on the sensor. In the next step concerning binding (step 8), the sensor containing the immobilized hPD1ex-H6F and the bound antibody-like molecule was immersed in an hCSF1R_His antigen solution at a concentration of 10 μg / ml. The signal amplification at this step is due to the presence of the FAB fragment for the hCSF1R_His antigen in the antibody-like molecule loaded onto the sensor.

[0170] The sensorgrams were analyzed using ForteBio Octet Data Analysis 9.0 software. The main results are shown in Table 5. The results clearly demonstrate that samples 08-003 and 08-004 exhibit co-binding to the hPD1ex-H6F antigen and the hCSF1R_His antigen.

[0171] [Table 5]

[0172] As a result, antibody-like molecules 08-003 and 08-004 are shown to bind simultaneously to the hPD1ex-H6F antigen and the hCSF1R_His antigen. Example 6. Analysis of the molecular weight of antibody-like molecules according to the present invention by reversed-phase ultra-high performance liquid chromatography (RP UHPLC) with mass spectrometric detection To confirm the correct assembly of the molecules, the molecular weights of the full-length antibody-like molecules according to the present invention were analyzed. This method allows the identification of full-length antibody-like molecules according to the present invention consisting of four distinct chains and the distinction of the antibody-like molecules from by-products formed from another set of chains. Together with the data from Examples 4 and 5, these data represent that a technical solution based on replacing a pair of constant domains CH1-CK with a pair consisting of β2-microglobulin of the human CD1b protein and a modified α3 domain results in the correct assembly of the antibody-like molecules according to the present invention.

[0173] The analysis was performed on a BioResolve Polyphenyl RP column (2.1×50 mm, particle size 2.7 μm; a BioResolve Polyphenyl RP pre-column, 2.1×5 mm, particle size 2.7 μm was also used) using an Agilent 1290 Infinity II UPLC-Agilent 6530 Q-Tof-mass spectrometry complex. Prior to analysis, all molecules were treated with PNGase F (Promega) enzyme to remove N-glycans. For this, the enzyme was diluted with water to a concentration of 1 unit of activity in 1 μl. A sample with a protein content of 50 μg was mixed with a buffer solution, the PNGase F solution was added at an enzyme:protein ratio of 1 unit:50 μg, and the mixture was incubated in a thermostat at (37.0±0.1)°C for 18 hours.

[0174] For analysis, 5 μg of the test solution was selected according to the measured protein concentration, and the concentration was adjusted to 0.2 mg / ml with mobile phase A. The sample injection volume was 5 μl. Prior to analysis, the chromatographic system was equilibrated for at least 30 minutes using a mobile phase with an initial ratio of mobile phase A: 95%, B: 5% (phase A contains 0.1% aqueous formic acid solution, and phase B contains 0.1% formic acid solution in isopropyl alcohol and 30% acetonitrile) until a stable pressure was obtained. The mass spectrometer was calibrated using a calibration standard solution according to the manufacturer's guidelines.

[0175] The sample was separated at a flow rate of 0.45 ml / min with a concentration gradient of mobile phase B (from 5% to 30% from 2 minutes to 3 minutes following sample introduction, then from 30% to 35% from 3 minutes to 8 minutes, and then from 35% to 95% from 8 minutes to 9 minutes) at a column temperature of (60 ± 1) °C; a chromatogram was obtained at a wavelength of 280 nm.

[0176] The data was processed with Protein Metrics. The results of mass spectrometry of the antibody-like molecules according to the present invention are shown in Table 6. In the table, each antibody-like molecule is provided together with the percentage of the mass corresponding to an accurately assembled antibody-like molecule consisting of four separate chains with respect to all the masses determined by the instrument.

[0177]

Table 6

[0178] The results of the analysis allow the conclusion that the masses of the full-sized antibody-like molecules consisting of four separate chains are dominant in all samples. Example 7. Confirmation of the accurate assembly of antibody-like molecules according to the present invention using proteolysis and mass spectrometry of the generated fragments To directly check the precise pairing of the light and heavy chains, antibody-like molecules according to the present invention were cleaved with a GingisKHAN protease (Genovis) whose recognition site is located in the hinge region of the antibody-like molecule (...KSCDK / THTCPPCP...). Such proteolysis resulted in the degradation of the full-length antibody-like molecule into Fc fragments, Fab fragments, and Fab-like fragments containing β2-microglobulin and modified α3 ​​domains of the human CD1b protein, with the CK and CH1 domains substituted. The fragments of the antibody-like molecule were analyzed following proteolysis using vertical electrophoresis under non-reducing conditions; the resulting fragments were also subjected to mass spectrometry.

[0179] The proteolytic reaction mixture contained 40 μg of antibody-like molecules and 40 units of GingisKHAN enzyme (at a ratio of 1 enzyme unit to 1 μg of protein) in a buffer consisting of 100 mM Tris-HCl, pH 8.0, and 1 mM cysteine, in a volume of 60 μl. The reaction was carried out for 1 hour in a thermostat at (37.0 ± 0.1) °C and stopped by adding iodoacetamide to a concentration of 10 mM. For electrophoresis, an SDS-containing buffer (maximum concentration of 1% SDS) was added to the sample to be produced, and SDS gel electrophoresis was performed under non-reducing conditions.

[0180] Figures 3-5 show the results of SDS gel electrophoresis. Following treatment of antibody-like molecules (08-001-08-006) with GingisKHAN protease (Figures 3 and 4), three main fragments were formed, which were observed in the 40-50 kDa region in the electrophoresis diagram and showed distinct mobilities in PAAG. A monospecific molecule containing a dimerization unit of the membrane-proximal domain of human CD1b protein and a variable fragment of the antibody prolgolimab (Figure 5, lane 4), as well as an IgG1-format monospecific antibody containing a variable fragment of the antibody prolgolimab, and an IgG1-format monospecific antibody containing a variable fragment of the antibody ocrelizumab (Figure 5, lanes 3 and 5) were also treated with GingisKHAN protease. Comparing the electrophoretic mobility of fragments formed during the proteolytic degradation of monospecific and bispecific antibody-like molecules, it was concluded that the fragments exhibiting the lowest mobility levels corresponded to Fc fragments, the fragments exhibiting moderate mobility levels corresponded to Fab fragments, and the fragments exhibiting the highest mobility levels corresponded to Fab-like fragments containing β2 microglobulin and modified α3 ​​domains of the CD1b protein, with substitutions of the CK and CH1 domains. In the case of antibody-like molecules 08-003 to 08-006, the electrophoretic maps showed further fragments formed as a result of nonspecific cleavage of the variable domain of the anti-CSF1R fragment. To determine the precise mass of the fragments formed as a result of proteolytic degradation, the inventors performed mass spectrometry analysis.

[0181] Prior to mass spectrometry analysis, immediately after adding iodoacetamide, the sample was transferred to a Zeba (MWCO 7kDa) column in 50 mM ammonium bicarbonate, pH (7.6±0.2), according to the manufacturer's guidelines; then, PNGase F was added to the sample at a protein ratio of 1 enzyme unit:50 μg, and the sample was thermostated at (37.0±0.1) °C for 18 hours.

[0182] Mass spectrometry of the generated fragments was performed as described in Example 6, with the following modifications: to obtain better peak resolution, the sample was separated at a column temperature of (60±1) °C with a mobile phase B concentration gradient (from 5% to 55% from 2 min to 20 min following sample introduction, then from 55% to 95% from 20 min to 21 min) at a flow rate of 0.4 ml / min; phase A contained 0.1% formic acid and 0.02% trifluoroacetic acid in water, and phase B contained a 0.1% formic acid solution in isopropyl alcohol and 30% acetonitrile).

[0183] Table 8 shows the results of RP UHPLC using mass spectrometry detection.

[0184] [Table 7]

[0185] The results show that all antibody-like molecules according to the present invention degrade, following proteolysis, into fragments corresponding in mass to an Fc fragment, a Fab fragment, and a Fab-like fragment containing β2 microglobulin and a modified α3 ​​domain of human CD1b protein, with the CK domain and CH1 domain substituted. This indicates that a technical solution based on substituting the constant domain CH1-CK pair with a pair consisting of β2 microglobulin and a modified α3 ​​domain of human CD1b protein results in the precise assembly of antibody-like molecules according to the present invention.

Claims

1. An antibody-like molecule that specifically binds to a first and a second target, wherein the antibody-like molecule is: 1) comprising a first antigen-binding fragment, and 2) comprising a second antigen-binding fragment, 1) The first antigen-binding fragment specifically binds to the first target and includes the following: a) The light chain of the first antigen-binding fragment, wherein the light chain of the first antigen-binding fragment comprises a light chain variable domain and a light chain constant domain; And, b) The heavy chain of the first antigen-binding fragment, wherein the heavy chain of the first antigen-binding fragment comprises a variable heavy chain domain of the antibody and a constant heavy chain domain of the antibody, wherein the constant heavy chain domain comprises a first (CH1) constant heavy chain domain and an Fc fragment monomer, wherein the Fc fragment monomer comprises a second (CH2) constant heavy chain domain and a third (CH3) constant heavy chain domain; and, 2) The second antigen-binding fragment specifically binds to the second target and comprises (i) or (ii) below: (i) a) The light chain of the second antigen-binding fragment, wherein the light chain of the second antigen-binding fragment comprises a light chain variable domain and a constant domain which is β2 microglobulin (β2M) of human CD1b (differentiation antigen group 1) protein having the amino acid sequence of SEQ ID NO: 1; And, b) The heavy chain of the second antigen-binding fragment, wherein the heavy chain of the second antigen-binding fragment comprises a heavy chain variable domain, a constant domain which is the α3 membrane proximal domain of the human CD1b protein having the amino acid sequence of SEQ ID NO: 2, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains; or, (ii) a) The light chain of the second antigen-binding fragment, wherein the light chain of the second antigen-binding fragment comprises a light chain variable domain and a constant domain which is the α3 membrane proximal domain of the human CD1b protein having the amino acid sequence of SEQ ID NO: 2; And, b) The heavy chain of the second antigen-binding fragment, wherein the heavy chain of the second antigen-binding fragment comprises a heavy chain variable domain, a constant domain which is β2 microglobulin (β2M) of the human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer comprising a second (CH2) and a third (CH3) heavy chain constant domain; Here, the β2 microglobulin (β2M) of the human CD1b protein having the amino acid sequence of SEQ ID NO: 1 and the α3 membrane proximal domain of the human CD1b protein having the amino acid sequence of SEQ ID NO: 2 form a heterodimer between them, stabilized by a disulfide bond. The aforementioned antibody-like molecule.

2. The CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are in contact with each other via a surface that has been modified to form the antibody-like molecule. Herein, the antibody-like molecule according to claim 1, wherein such modification in the CH3 domain of the heavy chain is a substitution that results in heterodimerization.

3. a) The CH3 domain of one heavy chain is modified such that, on the surface of the CH3 domain of one heavy chain, it comes into contact with the surface of the CH3 domain of the other heavy chain in the antibody-like molecule, and an amino acid residue is substituted with an amino acid residue having a larger side chain volume, resulting in the formation of a knob on the surface of the CH3 domain of one heavy chain that can fit into a hole on the surface of the CH3 domain of the other heavy chain. Furthermore, b) The CH3 domain of the other heavy chain is modified such that the surface of the CH3 domain of the second heavy chain is in contact with the surface of the CH3 domain of the first heavy chain in the antibody-like molecule, and an amino acid residue is substituted with an amino acid residue having a smaller side chain volume, resulting in the formation of a hole on the surface of the CH3 domain of the second heavy chain that can fit onto a knob on the interface of the CH3 domain of the first heavy chain; Here, the amino acid residue having a larger side chain volume is selected from the group including arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W), and here, the amino acid residue having a smaller side chain volume is selected from the group including alanine (A), serine (S), threonine (T), and valine (V). The antibody-like molecule according to claim 2.

4. The first light chain constant domain of the antibody-like molecule is selected from CK or CL. The antibody-like molecule according to claim 1.

5. The antibody-like molecule according to claim 1, wherein the CH3 domain of the antibody-like molecule is modified by introducing cysteine ​​(C) as an amino acid to the corresponding position of each CH3 domain so that a disulfide crosslink can be formed between both CH3 domains.

6. The CH3 domain of one heavy chain is modified to form a knob, and the CH3 domain of the other heavy chain is modified to form a hole, or vice versa. The antibody-like molecule according to claim 2 or 3.

7. The CH3 domain of one heavy chain has the amino acid substitution S354C / T366W according to the EU amino acid numbering scheme for antibodies, and the CH3 domain of the other heavy chain has the amino acid substitution Y349C / T366S / L368A / Y407V according to the EU amino acid numbering scheme for antibodies. The antibody-like molecule according to claim 6.

8. The CH3 domain of one heavy chain has the amino acid substitution Y349C / T366S / L368A / Y407 according to the EU amino acid numbering scheme for antibodies, and the CH3 domain of the other heavy chain has the amino acid substitution S354C / T366W according to the EU amino acid numbering scheme for antibodies. The antibody-like molecule according to claim 6.

9. The antibody-like molecule according to claim 1, wherein the Fc fragment belongs to IgG.

10. The antibody-like molecule according to claim 9, wherein the Fc fragment isotype is selected from the group: human IgG1, IgG2, or IgG4.

11. The antibody-like molecule according to claim 1, wherein the Fc fragment monomer includes substitutions that do not result in ADCC, CDC, and / or ADCP properties in the antibody-like molecule.

12. The antibody-like molecule according to claim 11, wherein the Fc fragment monomer includes substitutions of L234A and L235A according to the EU numbering scheme for antibody amino acids.

13. The antibody-like molecule according to claim 1, wherein the Fc fragment monomer includes a substitution that provides the sustained action of the antibody-like molecule.

14. The antibody-like molecule according to claim 13, wherein the Fc fragment monomer includes substitutions of M252Y, S254T, and T256E according to the EU numbering scheme for antibody amino acids.

15. The antibody-like molecule according to claim 1, wherein the Fc fragment monomer includes substitutions that result in enhanced ADCC, CDC, and / or ADCP properties in the antibody-like molecule.

16. The antibody-like molecule according to claim 15, wherein the Fc fragment monomer includes the substitution of E345R according to the EU numbering scheme for antibody amino acids.

17. The antibody-like molecule according to any one of claims 1 to 16, wherein the first and second targets can each be independently selected from the group comprising: CD20, BCMA, PD-1, PD-L1, CD47, GD2, AXL, TGF beta, CSF1R, blood coagulation factor 9 (FIX), blood coagulation factor 10 (FX), TNF alpha, IL17A, IL17F, or CD3.

18. The antibody-like molecule according to any one of claims 1 to 16, wherein the antibody-like molecule is a multispecific antibody-like molecule.

19. The antibody-like molecule is a bispecific, triplicate, quadruple, quintuple, hexaple, heptapoleque, octapleoleque, or nupleoleque antibody-like molecule. The antibody-like molecule according to any one of claims 1 to 16.

20. The aforementioned antibody-like molecule is a bivalent bispecific antibody-like molecule. The antibody-like molecule according to any one of claims 1 to 16.

21. An isolated nucleic acid encoding an antibody-like molecule according to any one of claims 1 to 20.

22. The isolated nucleic acid according to claim 21, wherein the nucleic acid is DNA.

23. An expression vector comprising the nucleic acid according to claim 21 or 22.

24. A method for producing host cells that produce antibody-like molecules according to any one of claims 1 to 20, comprising cell transformation by the vector according to claim 23.

25. A host cell for producing an antibody-like molecule according to any one of claims 1 to 20, comprising the nucleic acid according to claim 21 or 22.

26. A method for producing an antibody-like molecule according to claims 1 to 20: a) - Using an expression vector containing nucleic acid molecules encoding the light chain and heavy chain of the first antigen-binding fragment of the antibody-like molecule, - Using an expression vector containing nucleic acid molecules encoding the light and heavy chains of the second antigen-binding fragment of the antibody-like molecule, Steps to transform host cells, b) A step of culturing the host cells under conditions suitable for the synthesis of the antibody-like molecule; and, c) A step of isolating the antibody-like molecule from the cell culture, Methods that include...

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