Antibody-like molecules comprising heterodimers of human CD1b (cluster of differentiation 1) protein

Abstract

The present invention relates to the field of biotechnology, in particular to antibody-like molecules comprising heterodimers of the proximal domain of the human CD1b (Cluster of Differentiation 1) protein membrane, and to a method for producing said antibody-like molecules. The 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.

Description

Field of the Invention

[0001] The present invention relates to the field of biotechnology, and in particular to antibody-like molecules comprising heterodimers of the membrane-proximal domain of the human CD1b (cluster of differentiation 1) protein, and methods for producing the antibody-like molecules. The present invention further relates to nucleic acids encoding the antibody-like molecules, expression vectors, host cells for producing the antibody-like molecules, and methods for producing the cells. Background of the Invention Monoclonal antibodies in the form of chimeric, humanized or fully human molecules have been shown to be useful as effective drugs for the treatment of a variety of disorders or diseases.

[0003] Natural human antibody molecules are represented by heterotetramers 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 whole molecule form consist of bivalent ("two-armed") heterotetramers of heavy and light chains.

[0004] Diseases are often caused as a result of several pathologies and are accompanied by many comorbidities. Bispecific and multispecific antibodies are able to bind and thereby neutralize two or more different antigens per antibody molecule. The potential for significantly improved therapeutic properties (and value) of pharmaceutical products compared to monospecific monoclonal antibodies has made bispecific and multispecific antibodies an active area of ​​research. Over the past two decades, the literature has described many 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.

[0005] As mentioned above, there are many methods to generate molecules with combined antigen binding domains, i.e. molecules with antigen binding domains that are different from each other. However, each of these methods has its disadvantages.

[0006] Cross-linking by chemical methods is a time-consuming process because the corresponding moieties must be purified from homodimers and other undesirable byproducts. In addition, the chemical modification step can alter the integrity of the protein, thereby compromising its stability. Therefore, the above methods are generally ineffective and can lead to loss of antibody activity.

[0007] Cell fusion-based methods (e.g., hybridoma production) are random assembly of two heavy chains and two light chains, producing 10 antibody combinations. The target heteromultimeric antibodies are only a small fraction of the antibodies produced in this way. Isolation of the target heteromultimeric protein significantly reduces product yield and increases production costs.

[0008] Recombinant DNA technology is used to create a variety of heteromultimeric antibodies, such as single-chain Fv fragments without an Fc fragment and diabodies. The main disadvantage of this type of antibody molecule is the lack of an Fc domain, which results in the inability of the antibody to trigger effector functions (such as complement activation and binding to Fc receptors). Therefore, there is a need for bispecific or multispecific antibodies that contain a functional Fc domain.

[0009] Recombinant DNA technology is also used to design conjugated bispecific or multispecific antibodies using the Knob-into-Holes technique (see, for example, the disclosures of International Applications WO9627011 and WO9850431). One factor limiting the use of the above approach is that the light chains of the two initial antibodies should be identical when expressed in a single cell to prevent mispairing and formation of undesirable and / or inactive molecules.

[0010] The purity of a bispecific or multispecific antibody product depends on two factors: a) heterodimeric assembly of two different heavy chains co-expressed in cells, and b) Correct pairing of two different light chains with the cognate heavy chain.

[0011] The "knob-in-hole" technique for designing bispecific or multispecific antibodies solves the problem of correct heterodimer assembly of two different heavy chains co-expressed in cells. However, using only the knob-in-hole technique to design bispecific or multispecific antibodies only results in approximately 25% yield of correctly assembled bispecific or multispecific products, as the problem of correct pairing of the two different light chains with their cognate heavy chains remains unsolved.

[0012] The problem of correct pairing of two different light chains with their cognate heavy chains is solved in various ways as follows: 1. Use the same light chain in the first and second antigen-binding portions 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).

[0013] The disadvantage of the above solution is that it is not universal, because choosing a light chain that is suitable for both valencies can be problematic. In addition, when amino acid substitutions in the light chain are used to optimize the properties of the antigen-binding fragment, the substitutions will affect both valencies. Furthermore, the antibody binding to the second antigen may be disrupted.

[0014] 2. Use a single-chain format, ie, a format in which the light chain and the heavy chain of the antigen-binding fragment having specificity for the first antigen are linked to each other via a linker of several amino acids.

[0015] This format has technical disadvantages because it uses a linker to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to another binding protein (such as scFv or scFab), or to fuse, for example, the light and heavy variable domains (VH and VL) in scFv or the light chain (VL-CK (or CL)) to VH-CH1 in scFab. Linkers may cause problems in therapeutic settings. In fact, these foreign peptides can trigger an immune response against the linker itself or the junction area between the protein and the linker. In addition, the flexible nature of these peptides and their mobility make them more susceptible to proteolytic cleavage, which may lead to poor antibody stability, aggregation and increased immunogenicity.

[0016] 3. Modifying the CH1-CK domain in bispecific or multispecific antibodies allows for altering the interaction interface in bispecific or multispecific antibody expression techniques to preclude incorrect association of light chains. For example, international application WO2017059551 discloses various amino acid substitutions in CH1 and / or CK that promote preferred pairing between a desired heavy chain and a desired light chain.

[0017] Despite the various bispecific or multispecific antibody expression technologies described above, there is still a need in the art to improve the purity of bispecific or multispecific antibody products and scalable production solutions for producing correctly assembled bispecific or multispecific antibodies.

[0018] Invention Disclosure The novel format of antibody-like molecules comprising heterodimers of the membrane-proximal domain of the CD1b (Cluster of Differentiation 1) protein and the method for producing said antibody-like molecules developed by the authors of the present invention unexpectedly allow the production of high yields of products with correct heterodimeric assembly of two different heavy chains co-expressed in cells and correct pairing of two different light chains with the cognate heavy chain.

[0019] The novel format of antibody-like molecules comprising heterodimers of the membrane proximal domain of the CD1b protein and the method for producing said antibody-like molecules developed by the authors of the present invention unexpectedly allow the production of correctly assembled products of antibody-like molecules with high purity.

[0020] Thus, the above results reduce production costs and lead to a scalable production solution for properly assembled antibody-like molecules.

[0021] Definition and general approach Unless otherwise defined herein, all technical and scientific terms used in connection with the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art.

[0022] In addition, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, current classifications and methods for cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and hybridization and chemistry of proteins and nucleic acids as described herein are well known and widely used by those skilled in the art. Enzyme reactions and purification methods are performed according to manufacturer's guidelines, as is common in the art or as described herein.

[0023] The term "KD" in this specification refers to an affinity constant (or equilibrium constant), which is calculated from the ratio of Kd to Ka (ie, Kd / Ka) and is expressed as a molar concentration (M).

[0024] "Binding affinity" generally refers to the sum total strength of the 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 indicated, "binding affinity" refers to intrinsic (characteristic, true) binding affinity, which reflects a 1:1 interaction between binding pairs of members (e.g., an antibody and an antigen). The affinity of a molecule X for its binding partner Y can generally be represented by an affinity constant (KD). Preferred Kd values ​​are approximately 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 in this specification. Low-affinity antibodies generally bind antigen slowly and tend to dissociate, while high-affinity antibodies generally bind antigen faster and tend to remain in conjunction for a longer time. A variety of methods are known in the art for measuring binding affinity, any of which can be used for the purposes of the present invention.

[0025] The term "Kd," "koff," or "kdis" refers to the dissociation rate constant for a specific interaction between a binding molecule and an antigen. The dissociation rate constant, koff, can be measured using biofilm interferometry, for example, using the Octet™ system.

[0026] The term "Ka," "kon," or "on rate" refers to the association rate constant.

[0027] The term "R 2 ” refers to the coefficient of determination.

[0028] As used in this specification and the claims that follow, unless the context dictates otherwise, the words “include” and “comprise” or variations such as “includes”, “including”, “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0029] Antibody-like molecules The present invention relates to antibody-like molecules that specifically bind to a first and a second target.

[0030] The antibody-like molecules according to the present invention are monoclonal antibody-like molecules.

[0031] The term "monoclonal antibody-like molecule" refers to an antibody-like molecule that is synthesized and secreted by a single clonal cell population.

[0032] The antibody-like molecule according to the present invention is a recombinant antibody-like molecule.

[0033] The term "recombinant antibody-like molecule" refers to an antibody-like molecule expressed in a cell or cell line comprising a nucleotide sequence encoding the antibody-like molecule, wherein the nucleotide sequence is not associated with the cell in nature.

[0034] The antibody-like molecules according to the present invention are isolated antibody-like molecules.

[0035] The term "isolated" as used herein to describe various antibody-like molecules refers to an antibody-like molecule that has been identified and separated and / or regenerated from the cell or cell culture in which it is expressed. Impurities (contaminant components) from the natural environment are materials that generally interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteins or non-protein solutes. The isolated polypeptide is generally prepared by at least one purification step.

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

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

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

[0039] In one aspect, the invention relates to an antibody-like molecule that specifically binds to a first and a second target, wherein the antibody-like molecule comprises: 1) a first antigen-binding fragment that specifically binds to a first target and comprises: a) a light chain of the first antigen-binding fragment, wherein the light chain comprises a light chain variable domain and a light chain constant domain; and b) a heavy chain of a first antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain of an antibody and a heavy chain constant domain of an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; and 2) a second antigen-binding fragment that specifically binds to a second target and comprises: (i) a) a light chain of a second antigen-binding fragment, the light chain comprising a light chain variable domain and a light chain constant domain of β2 microglobulin (β2M), which is a human CD1b (cluster of differentiation 1) protein having the amino acid sequence of SEQ ID NO: 1; and b) a heavy chain of a second antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain, a constant domain that 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 second (CH2) and third (CH3) heavy chain constant domains; or (iI) a) a light chain of a second antigen-binding fragment, wherein the light chain comprises a light chain variable domain and a constant domain that is a human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; and b) a heavy chain of a second antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain, a constant domain of β2 microglobulin (β2M) which is a 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; 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 stabilized by a disulfide bond therebetween.

[0040] In the above antibody-like molecules that specifically bind to the first and second targets, the variable and constant domains are located in the heavy and light chains in the following sequence: 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) third (CH3) heavy chain constant domain; Light chain of the second antigen-binding fragment: 1) light chain variable domain, 2) the first human CD1b protein membrane proximal domain; Heavy chain of the second antigen-binding fragment: 1) heavy chain variable domain, 2) The second human CD1b protein membrane proximal domain, 3) the second (CH2) heavy chain constant domain, and 4) 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. 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 comprising 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 a first human CD1b protein membrane proximal domain. The second heavy chain comprises a heavy chain variable region (abbreviated as VH2 in this specification) and a heavy chain constant region comprising a second human CD1b protein membrane proximal domain and CH2-CH3.

[0042] The antibody-like molecules according to the present invention may be antibody-like molecules 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).

[0043] The VL and VH regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs), which are interspersed between more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen.

[0044] The constant regions of antibody-like molecules may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0045] As used in this specification, the term "antigen-binding fragment" refers to one or more fragments of an antibody-like molecule that retain the ability to specifically bind to an antigen.

[0046] "Kabat numbering scheme" or "numbering according to Kabat" as used in this application 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. NY Acad. Sci., 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0047] An antibody-like molecule of the present invention that "specifically binds" a target antigen or target is one that binds the antigen or target with sufficient affinity so that the antibody-like molecule can be used as a diagnostic and / or therapeutic agent targeting proteins or cells or tissues expressing the antigen, and has little cross-reactivity with other proteins.

[0048] The term "specifically binds to" a particular polypeptide or an epitope on a particular target polypeptide can be described by way of example by molecules having a Kd for the target 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.

[0049] In one embodiment, the term "specific binding" refers to binding wherein a molecule binds to a specific polypeptide or epitope on a specific polypeptide and does not substantially bind to any other polypeptide or epitope on a polypeptide.

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

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

[0052] CD1 (Cluster of Differentiation 1) refers to the Cluster of Differentiation 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. In a similar manner to MHC classes I and II, CD1 presents antigens for T cell recognition by interacting with T cell receptors. Unlike MHC classes I and II, the CD1 protein presents lipids and their derivatives rather than peptides.

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

[0054] The CD1 molecule is structurally similar to MHC class I. In a similar manner, a CD1 molecule is a non-covalent complex composed of two polypeptide chains: a polymorphic α chain (sometimes called the heavy chain) and a smaller, usually non-polymorphic chain called β2 microglobulin (also called the light chain).

[0055] The α chain forms an antigen-binding region comprising 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 the β2 microglobulin, forms a heterodimeric non-covalent complex. This heterodimeric non-covalent complex consisting of the CD1 α3 domain and the β2 microglobulin is referred to as a heterodimer of the CD1b protein membrane proximal domain in the present description.

[0056] The membrane proximal domain of the human CD1b protein is understood to mean the α3 domain of the human CD1b protein and the β2 microglobulin domain of the human CD1b protein.

[0057] The β2M of the human CD1b protein in the antibody-like molecule according to the present invention is the β2M of the wild-type human CD1b protein having the amino acid sequence of SEQ ID NO: 1.

[0058] The α3 membrane proximal domain of the human CD1b protein in the antibody-like molecule according to the present invention is a human CD1b protein α3 membrane proximal domain having an amino acid sequence of SEQ ID NO: 2, which differs from the wild-type human CD1b protein α3 membrane proximal domain having an amino acid sequence of SEQ ID NO: 3 in that it has S12K and N59D mutations and a GSC extension at the C-terminus.

[0059] In some embodiments of the invention, the antibody-like molecule comprises a CH3 domain of one heavy chain and a domain of the other heavy chain CH3 that are modified to form an antibody-like molecule in contact with each other via a surface, wherein the substitution in the heavy chain CH3 domain is modified to provide heterodimerization.

[0060] In some embodiments of the invention, the antibody-like molecule comprises: a) the CH3 domain of one heavy chain, which is modified such that, on the surface of the CH3 domain of one heavy chain that contacts the surface of the CH3 domain of the other heavy chain in the antibody-like molecule, the amino acid residues are substituted with amino acid residues 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 the hole on the surface of the CH3 domain of the other heavy chain, and b) the CH3 domain of the other heavy chain, which is modified such that, on the surface of the second heavy chain CH3 domain that contacts the surface of the first heavy chain CH3 domain in the antibody-like molecule, amino acid residues are substituted by amino acid residues with smaller side chain volume, resulting in the formation of a hole on the surface of the second heavy chain CH3 domain that is able to fit into the knob on the interface of the first heavy chain CH3 domain; wherein the amino acid residue with a larger side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W), and wherein the amino acid residue with a smaller side chain volume is selected from alanine (A), serine (S), threonine (T), and valine (V).

[0061] In some embodiments of the invention, the antibody-like molecule comprises the constant domain of the first light chain of an antibody, which is selected from CK or CL.

[0062] In some embodiments 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 into the corresponding position of each CH3 domain so that a disulfide bond can be formed between the two CH3 domains.

[0063] In some embodiments of the invention, the antibody-like molecule comprises a CH3 domain of one heavy chain that is modified to form a knob, and a CH3 domain of the other heavy chain that is modified to form a hole, or vice versa.

[0064] "Knob-in-hole" (knob-in-hole type interaction) is a method that makes it possible to avoid problems associated with mispairing byproducts. This method aims to force the pairing of two different heavy chains of an antibody or antibody-like molecule by introducing mutations into the CH3 domain to modify the contact interface. On one chain, bulky amino acids are replaced by amino acids with short side chains to create a "hole". Conversely, amino acids with larger side chains are introduced into the other CH3 domain to create a "knob". By co-expressing these two heavy chains, a high yield of heterodimer formation ("knob-in-hole") is generated relative to homodimer formation ("hole-in-hole" or "knob-in-hole") (see, for example, the disclosures of International Applications WO9627011 and WO9850431).

[0065] In some embodiments of the invention, the antibody-like molecule comprises a CH3 domain of one heavy chain having an S354C / T366W amino acid substitution according to the EU numbering scheme for antibody amino acids, and a CH3 domain of the other heavy chain having an Y349C / T366S / L368A / Y407V amino acid substitution according to the EU numbering scheme for antibody amino acids.

[0066] In some embodiments of the invention, the antibody-like molecule comprises a CH3 domain of one heavy chain having Y349C / T366S / L368A / Y407 amino acid substitutions according to the EU numbering scheme for antibody amino acids, and a CH3 domain of the other heavy chain having S354C / T366W amino acid substitutions according to the EU numbering scheme for antibody amino acids.

[0067] In some embodiments of the invention, the antibody-like molecule comprises an Fc fragment belonging to IgG.

[0068] In some embodiments of the invention, the antibody-like molecule comprises an Fc fragment selected from the group consisting of: human IgG1, IgG2, or IgG4.

[0069] In some embodiments of the invention, the antibody-like molecule comprises an Fc fragment monomer comprising substitutions that result in the absence of ADCC, CDC and / or ADCP properties in the antibody-like molecule.

[0070] In some embodiments of the invention, the antibody-like molecule comprises an Fc fragment monomer comprising L234A and L235A substitutions according to the EU numbering scheme for antibody amino acids.

[0071] In some embodiments of the invention, the antibody-like molecule comprises an Fc fragment monomer comprising substitutions that result in an elongated effect of the antibody-like molecule.

[0072] In some embodiments of the invention, the antibody-like molecule comprises an Fc fragment monomer comprising substitutions M252Y, S254T, and T256E according to the EU numbering scheme for antibody amino acids.

[0073] In some embodiments of the invention, the antibody-like molecule comprises an Fc fragment monomer comprising substitutions that result in enhanced ADCC, CDC and / or ADCP properties in the antibody-like molecule.

[0074] In some embodiments of the invention, the antibody-like molecule comprises an Fc fragment monomer comprising an E345R substitution according to the EU numbering scheme for antibody amino acids.

[0075] The above mutations in the Fc fragment are numbered according to the EU numbering scheme for antibody chain amino acids (Edelman, GM et al., Proc. Natl. Acad. Sci. USA 63 (1969), pp. 78-85; Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991)).

[0076] The term "effector function" of an antibody or antibody-like molecule refers to the biological activity attributable to the Fc region (native Fc region sequence or Fc region amino acid variant) of the antibody or antibody-like molecule, 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 receptor, BCR) and B cell activation.

[0077] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies or antibody-like molecules on target cells and subsequently cause lysis or phagocytosis of the target cell.

[0078] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the 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; PBMCs and NK cells are preferred.

[0079] "Complement-dependent cytotoxicity" and "CDC" refer to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (CIq) to a molecule complexed with a cognate antigen.

[0080] In some embodiments of the invention, the antibody-like molecule specifically binds to a first and a second target, wherein the target is a single antigen.

[0081] In some embodiments of the invention, the antibody-like molecule specifically binds to a first and a second target, where target is understood to mean two, three or more structurally similar antigens.

[0082] Those skilled in the art will appreciate that antibodies and antibody-like molecules can have cross-reactivity due to similarities in antigen / target protein structure. For example, antibodies directed against programmed cell death protein 1 (PD-1) may also have cross-reactivity to other members of the CD28 receptor family, including CD28, CTLA-4, ICOS, and BTLA. In turn, antibodies directed against GITR (glucocorticoid-induced TNFR-related protein) may also have cross-reactivity to other representatives of the TNFRSF superfamily, including CD137, OX40, and CD27. Cross-reactivity of therapeutic antibodies is generally avoided. For example, PCT application WO 2006 / 121168 discloses antibodies that bind to PD-1 but do not substantially bind to CD28, CTLA-4, and human ICOS. However, cross-reactivity is an important feature of many therapeutic antibodies; for example, the prior art provides the monoclonal antibody ustekinumab (Juliane Weber et al., Ustekinumab, BioDrugs, 2009, 23(1):53-61. doi: 10.2165 / 00063030-200923010-00006) that binds to the p40 subunit common to both IL12 and IL23. Antibodies comprising two antigen-binding fragments are also known, wherein one of the antigen-binding fragments binds to two antigens, IL17A or IL17F, because these interleukins have a high percentage of structural identity; therefore, these antibodies comprising two antigen-binding fragments are simultaneously specific for three antigens. For example, patent US10562967 provides an antibody comprising two antigen-binding fragments and having specificity for three antigens IL-23p19, IL-17A and IL-17F, while the disclosure of PCT application WO2017188850 provides an antibody comprising two antigen-binding fragments and having specificity for three antigens TNFα, IL-17A and IL-17F. Based on the above, the target bound by the antibody-like molecule according to the present invention is understood to mean an antigen (protein or other property) comprising an epitope recognized and bound by the antigen-binding site of the antibody-like molecule, or an antigen (protein or other property) comprising an epitope recognized and bound by the antigen-binding site of the antibody-like molecule. In the case where the antibody-like molecule comprises multiple antigen-binding sites that recognize and bind to different targets (including the case where a single antigen-binding site is capable of recognizing and binding to multiple antigens), such an antibody or antibody-like molecule is considered to be multispecific (bispecific in the case of two antigens, trispecific in the case of three antigens, tetraspecific in the case of four antigens, etc.).

[0083] Antibody-like molecules according to the invention which comprise two antigen-binding fragments and which specifically bind to at least three antigens, such as three, four, five, six, seven, eight, nine, etc. antigens are within the scope of the invention.

[0084] In some embodiments of the present invention, the antibody-like molecule specifically binds to a first and a second target, wherein the first and the second target can each be independently selected from: CD20, BCMA, PD-1, PD-L1, CD47, GD2, AXL, TGFβ, CSF1R, coagulation factor 9 (FIX), coagulation factor 10 (FX), TNFα, IL17A, IL17F or CD3.

[0085] In some embodiments of the invention, the antibody-like molecule is a multispecific antibody-like molecule.

[0086] In some embodiments of the invention, the antibody-like molecule is a bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, heptaspecific, octaspecific, or nonaspecific antibody-like molecule.

[0087] In some embodiments, the antibody-like molecule is a bivalent bispecific antibody-like molecule.

[0088] The present application materials provide the following antibody-like molecules: 08-001, 08-002, 08-003, 08-004, 08-005 or 08-006.

[0089] 08-001 is an antibody-like molecule that specifically binds to PD1 and CD20 and contains the following: 1) A first antigen-binding fragment that specifically binds to PD1 and comprises: a) a light chain of a first antigen-binding fragment, the light chain of which comprises a light chain variable domain (Palolimab_VL) and a light chain constant domain of the antibody Prolgolimab having SEQ ID NO: 5; and b) a heavy chain of a first antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain of the antibody palolizumab having SEQ ID NO: 4 (palolizumab_VH) and an antibody heavy chain constant domain comprising a first (CH1) heavy chain constant domain and an Fc fragment monomer comprising second (CH2) and third (CH3) heavy chain constant domains; and 2) a second antigen-binding fragment that specifically binds to CD20 and comprises: a) a light chain of a second antigen-binding fragment, the light chain of which comprises a light chain variable domain (ocrelizumab_VL) of the antibody ocrelizumab having the amino acid sequence of SEQ ID NO: 7 and a constant domain that is a human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; and b) the heavy chain of the second antigen-binding fragment, the heavy chain of which comprises the heavy chain variable domain of the antibody ocrelizumab having SEQ ID NO: 6 (ocrelizumab_VH), the constant domain of the human CD1b protein β2 microglobulin (β2M) having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains.

[0090] 08-001 is an antibody-like molecule that specifically binds to PD1 and CD20 and contains the following: 1) A first antigen-binding fragment that specifically binds to PD1 and comprises: a) the light chain of the first antigenic fragment having the amino acid sequence of SEQ ID NO: 11 (Paloclimab_VL_CK); and b) the heavy chain of the first antigen-binding fragment having the amino acid sequence of SEQ ID NO: 10 (Paloclimab_VH_HC_hole); and 2) a second antigen-binding fragment that specifically binds to CD20 and comprises: a) a light chain of a 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 (ocrelizumab_VH_b2m_Fc_knob) having the amino acid sequence of SEQ ID NO: 12.

[0091] 08-002 is an antibody-like molecule that specifically binds to CD20 and PD1 and comprises: 1) A first antigen-binding fragment that specifically binds to CD20 and comprises: a) a light chain of a first antigen-binding fragment, the light chain of which comprises a light chain variable domain (ocrelizumab_VL) and a light chain constant domain of the antibody ocrelizumab having SEQ ID NO: 7; and b) the heavy chain of a first antigen-binding fragment, the heavy chain of which comprises the heavy chain variable domain of the antibody ocrelizumab having SEQ ID NO: 6 (ocrelizumab_VH) and the antibody heavy chain constant domain comprising a first (CH1) heavy chain constant domain and an Fc fragment monomer comprising the first (CH1) heavy chain constant domain and comprising second (CH2) and third (CH3) heavy chain constant domains; and 2) A second antigen-binding fragment that specifically binds to PD1 and comprises: a) a light chain of a second antigen-binding fragment, the light chain of which comprises a light chain variable domain of the antibody palolizumab having the amino acid sequence of SEQ ID NO: 5 (palolizumab_VL) and a constant domain that is a human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; and b) the heavy chain of the second antigen-binding fragment, the heavy chain of which comprises the heavy chain variable domain of the antibody palolizumab having SEQ ID NO: 4 (ocrelizumab_VH), the constant domain of β2 microglobulin (β2M) which is the human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains.

[0092] 08-002 is an antibody-like molecule that specifically binds to CD20 and PD1 and comprises: 1) A first antigen-binding fragment that specifically binds to CD20 and comprises: 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) A second antigen-binding fragment that specifically binds to PD1 and comprises: a) a light chain of a second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 17 (Paloclimab_VL_CD1b); and b) The heavy chain of the second antigen-binding fragment (Paloclimumab_VH_b2m_Fc_knob) having the amino acid sequence of SEQ ID NO: 16.

[0093] 08-003 is an antibody-like molecule that specifically binds to PD1 and CSF1R and comprises: 1) A first antigen-binding fragment that specifically binds to PD1 and comprises: a) a light chain of a first antigen-binding fragment, the light chain comprising a light chain variable domain (Paloclimab_VL) and a light chain constant domain of the antibody palolizumab having SEQ ID NO: 5; and b) a heavy chain of a first antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain of the antibody palolizumab having SEQ ID NO: 4 (palolizumab_VH) and an antibody heavy chain constant domain comprising a first (CH1) heavy chain constant domain and an Fc fragment monomer comprising second (CH2) and third (CH3) heavy chain constant domains; and 2) a second antigen-binding fragment that specifically binds to CSF1R and comprises: a) a light chain of a second antigen-binding fragment, the light chain of which comprises a light chain variable domain of an antibody against CSF1R (anti-CSF1R_VL) having SEQ ID NO: 9 and a constant domain that is a human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; and b) the heavy chain of the second antigen-binding fragment, which comprises the heavy chain variable domain of an antibody against CSF1R having SEQ ID NO: 8 (anti-CSF1R_VH), the constant domain of β2 microglobulin (β2M) which is the human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains.

[0094] 08-003 is an antibody-like molecule that specifically binds to PD1 and CSF1R and comprises: 1) A first antigen-binding fragment that specifically binds to PD1 and comprises: a) the light chain of the first antigen-binding fragment (Paloclimab_VL_CK) having the amino acid sequence of SEQ ID NO: 11; and b) the heavy chain of the first antigen-binding fragment (Paloclimumab_VH_HC_hole) having the amino acid sequence of SEQ ID NO: 10; and 2) a second antigen-binding fragment that specifically binds to CSF1R and comprises: a) a light chain of a 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 (anti-CSF1R_VH_b2m_Fc_knob) having the amino acid sequence of SEQ ID NO: 18.

[0095] 08-004 is an antibody-like molecule that specifically binds to CSF1R and PD1 and comprises: 1) A first antigen-binding fragment that specifically binds to CSF1R and comprises: a) a light chain of a first antigen-binding fragment, wherein the light chain comprises a light chain variable domain (anti-CSF1R_VL) of an antibody against CSF1R having SEQ ID NO: 9 and a light chain constant domain; and b) a heavy chain of a first antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain of an antibody against CSF1R (anti-CSF1R_VH) having SEQ ID NO: 8 and an antibody heavy chain constant domain comprising a first (CH1) heavy chain constant domain and an Fc fragment monomer comprising second (CH2) and third (CH3) heavy chain constant domains; and 2) A second antigen-binding fragment that specifically binds to PD1 and comprises: a) a light chain of a second antigen-binding fragment, the light chain of which comprises a light chain variable domain of the antibody palolizumab having the amino acid sequence of SEQ ID NO: 5 (palolizumab_VL) and a constant domain that is a human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; and b) the heavy chain of the second antigen-binding fragment, the heavy chain of which comprises the heavy chain variable domain of the antibody palolizumab having SEQ ID NO: 4 (palolizumab_VH), the constant domain of β2 microglobulin (β2M) which is the human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains.

[0096] 08-004 is an antibody-like molecule that specifically binds to CSF1R and PD1 and comprises: 1) A first antigen-binding fragment that specifically binds to CSF1R and comprises: 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 SEQ ID NO: 20 (anti-CSF1R_VH_HC_hole); and 2) A second antigen-binding fragment that specifically binds to PD1 and comprises: a) a light chain of a second antigen-binding fragment having the amino acid sequence of SEQ ID NO: 17 (Paloclimab_VL_CD1b); and b) The heavy chain of the second antigen-binding fragment (Paloclimumab_VH_b2m_Fc_knob) having the amino acid sequence of SEQ ID NO: 16.

[0097] 08-005 is an antibody-like molecule that specifically binds to CD20 and CSF1R and comprises: 1) A first antigen-binding fragment that specifically binds to CD20 and comprises: a) a light chain of a first antigen-binding fragment, the light chain of which comprises a light chain variable domain (ocrelizumab_VL) and a light chain constant domain of the antibody ocrelizumab having SEQ ID NO: 7; and b) a heavy chain of a first antigen-binding fragment, the heavy chain of which comprises the heavy chain variable domain of the antibody ocrelizumab having SEQ ID NO: 6 (ocrelizumab_VH) and an antibody heavy chain constant domain comprising a first (CH1) heavy chain constant domain and an Fc fragment monomer comprising second (CH2) and third (CH3) heavy chain constant domains; and 2) a second antigen-binding fragment that specifically binds to CSF1R and comprises: a) a light chain of a second antigen-binding fragment, the light chain of which comprises a light chain variable domain of an antibody against CSF1R (anti-CSF1R_VL) having SEQ ID NO: 9 and a constant domain that is a human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; and b) the heavy chain of the second antigen-binding fragment, which comprises the heavy chain variable domain of an antibody against CSF1R having SEQ ID NO: 8 (anti-CSF1R_VH), the constant domain of β2 microglobulin (β2M) which is the human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains.

[0098] 08-005 is an antibody-like molecule that specifically binds to CD20 and CSF1R and comprises: 1) A first antigen-binding fragment that specifically binds to CD20 and comprises: 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) a second antigen-binding fragment that specifically binds to CSF1R and comprises: a) a light chain of a 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 (anti-CSF1R_VH_b2m_Fc_knob) having the amino acid sequence of SEQ ID NO: 18.

[0099] 08-006 is an antibody-like molecule that specifically binds to CSF1R and CD20 and comprises: 1) A first antigen-binding fragment that specifically binds to CSF1R and comprises: a) a light chain of a first antigen-binding fragment, wherein the light chain comprises a light chain variable domain (anti-CSF1R_VL) of an antibody against CSF1R having SEQ ID NO: 9 and a light chain constant domain; and b) a heavy chain of a first antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain of an antibody against CSF1R (anti-CSF1R_VH) having SEQ ID NO: 8 and an antibody heavy chain constant domain comprising a first (CH1) heavy chain constant domain and an Fc fragment monomer comprising second (CH2) and third (CH3) heavy chain constant domains; and 2) a second antigen-binding fragment that specifically binds to CD20 and comprises: a) a light chain of a second antigen-binding fragment, the light chain of which comprises the light chain variable domain of the antibody ocrelizumab having the amino acid sequence of SEQ ID NO: 7 (ocrelizumab_VH) and a constant domain that is the human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; and b) the heavy chain of the second antigen-binding fragment, the heavy chain of which comprises the heavy chain variable domain of the antibody ocrelizumab having SEQ ID NO: 6 (ocrelizumab_VH), the constant structure of β2 microglobulin (β2M) which is the human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains.

[0100] 08-006 is an antibody-like molecule that specifically binds to CSF1R and CD20 and comprises: 1) A first antigen-binding fragment that specifically binds to CSF1R and comprises: 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 SEQ ID NO: 20 (anti-CSF1R_VH_HC_hole); and 2) a second antigen-binding fragment that specifically binds to CD20 and comprises: a) a light chain of a 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 (ocrelizumab_VH_b2m_Fc_knob) having the amino acid sequence of SEQ ID NO: 12.

[0101] These antibody-like molecules are given for illustrative purposes to demonstrate the operability of the antibody-like molecule format according to the present invention and its unexpected properties. Such antibody-like molecules should not be interpreted as limiting the antibody-like molecules of the present invention in any way.

[0102] The yield parameters of products with correct heterodimeric assembly of two different heavy chains and correct pairing between two different light chains and the cognate heavy chain are independent of the heavy and light chain variable fragments of the antibody-like molecule and their specificity for the antigen.

[0103] The antibody-like molecules according to the invention can be used to treat various diseases, in particular oncological diseases, autoimmune diseases or diseases associated with coagulation (clotting) disorders.

[0104] Nucleic acid molecules In one aspect, the present invention relates to nucleic acids encoding the above antibody-like molecules.

[0105] The terms "nucleic acid," "nucleic sequence," "nucleic acid sequence," "polynucleotide," "oligonucleotide," "polynucleotide sequence," and "nucleotide sequence" used interchangeably in this specification refer to a precise sequence of nucleotides, whether modified or not, that defines a segment or region of a nucleic acid, whether or not containing non-natural nucleotides, and that is double-stranded DNA or RNA, single-stranded DNA or RNA, or the transcription product of said DNA.

[0106] Unless otherwise indicated, the term nucleotide sequence includes its complement. Thus, a nucleic acid having a specific sequence should be understood to include a nucleic acid having its complementary strand having its complementary sequence.

[0107] In any of the described embodiments, the nucleic acid molecule can be isolated.

[0108] An "isolated" nucleic acid molecule is one that has been identified and separated from at least one nucleic acid molecule impurity. An isolated nucleic acid molecule is different from the form or collection in which it is found under natural conditions. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecule that exists in a cell under natural conditions.

[0109] 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 NO: 22 to 33. The nucleic acid molecule may also comprise any combination of said nucleotide sequences.

[0110] In some embodiments of the invention, the isolated nucleic acid is DNA.

[0111] In one embodiment, the present invention relates to a nucleic acid molecule encoding the light chain or heavy chain amino acid sequence of the above antibody-like molecule according to the present invention, which is selected from: - a nucleic acid encoding the amino acid sequence of the light chain of the first antigen-binding fragment, the light chain comprising a light chain variable domain and a light chain constant domain; - a nucleic acid encoding the amino acid sequence of the heavy chain of a first antigen-binding fragment, the heavy chain of which comprises the heavy chain variable domain of an antibody and the heavy chain constant domain of an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; - a nucleic acid encoding the amino acid sequence of the light chain of the second antigen-binding fragment, the light chain of which comprises a light chain variable domain and the constant domain of β2 microglobulin (β2M), which is a human CD1b (cluster of differentiation 1) protein having the amino acid sequence of SEQ ID NO: 1; - a nucleic acid encoding the amino acid sequence of the heavy chain of a second antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain, a constant domain that 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; - a nucleic acid encoding the amino acid sequence of the light chain of the second antigen-binding fragment, wherein the light chain comprises a light chain variable domain and a constant domain that is the human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; or - A nucleic acid encoding the amino acid sequence of the heavy chain of the second antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain, a constant domain of β2 microglobulin (β2M), which is a human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains.

[0112] The nucleic acid molecule may also comprise a combination of the above nucleotide sequences necessary to produce the antibody-like molecule according to the present invention.

[0113] As will be appreciated by those skilled in the art, due to the redundancy of genetic code, a variety of different DNA sequences can encode the amino acid sequence of the light chain or heavy chain or its fragment (VH, VL, CDR etc.) of antibody-like molecule according to the present invention.Creating these alternative DNA sequences encoding the same amino acid sequence is fully within the skill of trained personnel in this area.This type of variant DNA sequence is within the scope of the present invention.

[0114] The nucleic acid molecules according to the invention may be isolated from any source which produces the antibody-like molecules according to the invention.In certain embodiments of the invention, the nucleic acid molecules of the invention may be synthesized by chemical synthesis rather than isolated.

[0115] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the heavy chain amino acid sequence (Paloclimumab_VH_HC_H) of the first antigen-binding fragment of antibody-like molecules 08-001 and 08-003 and comprises a nucleotide sequence having SEQ ID NO: 22.

[0116] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the light chain amino acid sequence (Paloclimab_VL_CK) of the first antigen-binding fragment of antibody-like molecules 08-001 and 08-003 and comprises a nucleotide sequence having SEQ ID NO: 23.

[0117] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the heavy chain amino acid sequence of the second antigen-binding fragment of antibody-like molecules 08-001 and 08-006 (ocrelizumab_VH_b2m_Fc_knob) and comprises a nucleotide sequence having SEQ ID NO: 24.

[0118] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the light chain amino acid sequence (ocrelizumab_VL_CD1b) of the second antigen-binding fragment of antibody-like molecules 08-001 and 08-006 and comprises a nucleotide sequence having SEQ ID NO: 25.

[0119] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the heavy chain amino acid sequence (ocrelizumab_VL_CD1b) of the first antigen-binding fragment of antibody-like molecules 08-002 and 08-005 and comprises a nucleotide sequence having SEQ ID NO: 26.

[0120] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the light chain amino acid sequence (ocrelizumab_VL_CK) of the first antigen-binding fragment of antibody-like molecules 08-002 and 08-005 and comprises a nucleotide sequence having SEQ ID NO: 27.

[0121] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the heavy chain amino acid sequence (Paloclimumab_VH_b2m_Fc_knob) of the second antigen-binding fragment of antibody-like molecules 08-002 and 08-004 and comprises a nucleotide sequence having SEQ ID NO: 28.

[0122] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the light chain amino acid sequence (Paloclimumab_VL_CD1b) of the second antigen-binding fragment of antibody-like molecules 08-002 and 08-004 and comprises a nucleotide sequence having SEQ ID NO: 29.

[0123] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the heavy chain amino acid sequence of the second antigen-binding fragment of antibody-like molecules 08-003 and 08-005 (anti-CSF1R_VH_b2m_Fc_knob) and comprises a nucleotide sequence having SEQ ID NO: 30.

[0124] In some embodiments of the present invention, the nucleic acid encodes the light chain amino acid sequence (anti-CSF1R_VL_CD1b) of the second antigen-binding fragment of antibody-like molecules 08-003 and 08-005 and comprises a nucleotide sequence having SEQ ID NO: 31.

[0125] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the heavy chain amino acid sequence (anti-CSF1R_VH_HC_hole) of the first antigen-binding fragment of antibody-like molecules 08-004 and 08-006 and comprises a nucleotide sequence having SEQ ID NO: 32.

[0126] In some embodiments of the present invention, the nucleic acid is a nucleic acid encoding the light chain amino acid sequence (anti-CSF1R_VL_CK) of the first antigen-binding fragment of antibody-like molecules 08-004 and 08-006 and comprises a nucleotide sequence having SEQ ID NO: 33.

[0127] Nucleic acid molecules can be used to express antibody-like molecules according to the present invention.

[0128] carrier In one aspect, the present invention relates to an expression vector comprising any of the above nucleic acid molecules encoding the corresponding amino acid sequence of the antibody-like molecule according to the present invention.The present invention relates to a vector suitable for expressing any of the nucleotide sequences described herein.

[0129] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0130] As used in this specification, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0131] The present invention relates to a vector comprising a nucleic acid molecule encoding any one of the above antibody-like molecules or a structural portion thereof selected from the following: - a nucleic acid encoding the amino acid sequence of the light chain of the first antigen-binding fragment, the light chain comprising a light chain variable domain and a light chain constant domain; - a nucleic acid encoding the amino acid sequence of the heavy chain of a first antigen-binding fragment, the heavy chain of which comprises the heavy chain variable domain of an antibody and the heavy chain constant domain of an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; - a nucleic acid encoding the amino acid sequence of the light chain of the second antigen-binding fragment, the light chain of which comprises a light chain variable domain and the constant domain of β2 microglobulin (β2M), which is a human CD1b (cluster of differentiation 1) protein having the amino acid sequence of SEQ ID NO: 1; - a nucleic acid encoding the amino acid sequence of the heavy chain of a second antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain, a constant domain that 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; - a nucleic acid encoding the amino acid sequence of the light chain of the second antigen-binding fragment, wherein the light chain comprises a light chain variable domain and a constant domain that is the human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; or - A nucleic acid encoding the amino acid sequence of the heavy chain of the second antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain, a constant domain of β2 microglobulin (β2M), which is a human CD1b protein having the amino acid sequence of SEQ ID NO: 1, and an Fc fragment monomer comprising the second (CH2) and third (CH3) heavy chain constant domains.

[0132] In some embodiments of the invention, the vector is a plasmid, a circular double-stranded DNA fragment into which additional DNA segments can be inserted.

[0133] In some embodiments of the invention, the vector is a viral (expression) vector, wherein additional DNA segments can be inserted into the viral genome.

[0134] In some embodiments of the present invention, carrier can be introduced therein to their host cell and replicate autonomously (for example, bacterial vector and episomal vector with bacterial replication origin).In a further embodiment of the present invention, carrier (for example non-episomal vector) can be integrated into the genome of host cell after being introduced into host cell, and replicates together with host gene thus.In addition, some carrier can instruct the expression of the gene that they are operably connected.This type of carrier is referred to as " recombinant expression vector " (or simply referred to as " expression vector ") in this article.

[0135] In some embodiments of the present invention, expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, and the like. DNA molecules can be inserted into vectors so that the transcriptional and translational control sequences within the vectors exert their intended functions of regulating DNA transcription and translation. Expression vectors and expression control sequences can be selected to be compatible with the host cell used for expression.

[0136] In some embodiments of the invention, DNA molecules encoding part or all of the heavy and light chain sequences may be inserted into separate vectors.

[0137] In one embodiment, any combination of the above DNA molecules are introduced into the same expression vector.

[0138] In one embodiment of the invention, the DNA molecule can be introduced into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the gene segments of the antibody or antibody-like molecule and the vector, or blunt end ligation if no restriction sites are present).

[0139] In some embodiments of the present invention, suitable vectors are vectors that include restriction sites so that any VH or VL sequence can be easily inserted and expressed, as described above. The recombinant expression vector can also encode a signal peptide that promotes secretion of the chain of the antibody-like molecule from the host cell. The gene of the antibody-like molecule chain can be cloned into the vector so that the signal peptide is in-frame connected to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0140] In some embodiments of the present invention, the vector may include an expression control sequence. As used in this specification, the term "expression control sequence" refers to a polynucleotide sequence necessary for achieving expression and processing of the coding sequence into which it is inserted. Those skilled in the art will appreciate that the design of the expression vector, including the selection of expression control sequences, may depend on factors such as the choice of the host cell type to be transformed, the desired expression level of the antibody or antibody-like molecule, and the like. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that improve protein stability; and sequences that enhance protein secretion when desired. The nature of such expression control sequences varies depending on the host organism; in prokaryotes, such expression control sequences generally include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such expression control sequences generally include promoters and transcription termination sequences. Preferred expression control sequences for mammalian expression host cells include viral elements that ensure high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (such as CMV promoter / enhancer), simian virus 40 (SV40) (such as SV40 promoter / enhancer), adenovirus (e.g., major late promoter adenovirus (AdMLP)), polyoma virus, and strong mammalian promoters such as TTR promoter, natural immunoglobulin promoter, or actin promoter. Expression control sequences include at least all components whose presence is important for expression and processing.

[0141] In some embodiments of the present invention, in addition to the antibody-like molecule chain gene and expression control sequence, the recombinant expression vector of the present invention may also carry additional sequences, such as sequences that regulate the replication of the vector in host cells (e.g., origins of replication) and selective marker genes. The selective marker gene promotes the selection of host cells into which the vector has been introduced.

[0142] host cells In one aspect, the present invention relates to a method for producing a host cell to produce any one of the above antibody-like molecules according to the present invention, and comprises transforming the cell with one or more of the above vectors comprising a combination of the above nucleotide sequences necessary for producing the above antibody-like molecules according to the present invention.

[0143] In one aspect, the present invention relates to a host cell producing any one of the above antibody-like molecules according to the present invention, comprising a combination of the above nucleotide sequences necessary for producing the above antibody-like molecules according to the present invention.

[0144] As used herein, the term "host cell" refers to a cell into which a recombinant expression vector is introduced. The present invention relates to a host cell, which may include, for example, the vector according to the present invention described above. The present invention further relates to a host cell comprising, 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 specific subject cell, but also to the progeny of this cell. Since modifications may occur in progeny due to mutations or environmental influences, such progeny may not, in fact, be identical to the parent cell; however, such cells are still included within the scope of the term "host cell" as used herein.

[0145] The nucleic acid molecules of antibody-like molecules according to the present invention and the carriers that comprise these nucleic acid molecules can be used for transfection mammalian cells, vegetable cells, bacterial cells or yeast cells.Transfection can be carried out by any known method that is used for polynucleotide to be introduced into host cells.It is well known in the art that the method that is used for heterologous polynucleotide to be introduced into mammalian cells, and comprises transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, polynucleotide to be encapsulated in liposome and DNA is directly microinjected in nucleus of glucan mediation.In addition, nucleic acid molecules can be introduced into mammalian cells by viral (expression) vector.

[0146] The mammalian cell line used as the host for conversion is well known in the art, and comprises available multiple immortalized cell lines.These comprise for example Chinese hamster ovary (CHO) cell, NSO cell, SP2 cell, HEK-293T cell, FreeStyle 293 cell (Invitrogen), NIH-3T3 cell, HeLa cell, baby hamster kidney (BHK) cell, African green monkey kidney cell (COS), human hepatocellular carcinoma cell (for example Hep G2), A549, SK-HEP1, HUH7, Hep-RG cell and many other cell lines.By determining which cell line has high expression level and the necessary characteristics of produced protein are provided to select cell line.Operable other cell lines are insect cell lines, such as Sf9 or Sf21 cells.When the recombinant expression vector of the above antibody-like molecule of the present invention of encoding is incorporated into mammalian host cell, by cultivating host cell, be enough to the time period of expressing antibody-like molecule of the present invention in host cell, or more preferably, antibody-like molecule is secreted into the culture medium of wherein cultivating host cell and produces antibody-like molecule. The antibody-like molecules of the present invention can be isolated from the culture medium using standard protein purification techniques. Plant host cells include, for example, tobacco, Arabidopsis thaliana, duckweed, corn, wheat, potato, etc. Bacterial host cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0147] In addition, various known techniques can be used to increase the level of production of the above antibody-like molecules of the present invention from production cell lines. For example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions.

[0148] It is possible that the above antibody-like molecules of the present invention will have glycosylation patterns that differ from one another in different cell lines. However, the above antibody-like molecules of the present invention encoded by nucleic acid molecules described herein or comprising an amino acid sequence provided herein are part of the present invention and have nothing to do with the glycosylation of the binding molecule and are generally unrelated to the presence or absence of post-translational modifications.

[0149] The above host cells are not related to host cells generated using human embryos.

[0150] The above host cells are not related to host cells generated by modifying the genetic integrity of human germline cells.

[0151] Methods for producing antibody-like molecules In one aspect, the present invention relates to a method for producing any one of the above antibody-like molecules, wherein the method comprises the following steps: a) Transform the host cell with - an expression vector comprising a nucleic acid molecule encoding the light chain and the heavy chain of the first antigen-binding fragment of the antibody-like molecule, - an expression vector comprising a nucleic acid molecule encoding the light chain and the heavy chain of the second antigen-binding fragment of the antibody-like molecule, b) culturing the host cell under conditions suitable for the synthesis of said antibody-like molecule; and c) isolating the antibody-like molecule from the cell culture.

[0152] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The electrophoresis diagram of the antibody-like molecule sample after purification from culture medium. 7.5% PAAG, non-reducing conditions.

[0153] M - standard marker for Precision Plus Protein™ Dual Color Standard (BIO-RAD), the molecular weight of the corresponding lane is indicated in 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.

[0154] Figure 2 Electrophoresis of antibody-like molecules purified from culture medium. 12% PAAG, reducing conditions.

[0155] M - standard marker for Precision Plus Protein™ Dual Color Standard (BIO-RAD), the molecular weight of the corresponding lane is indicated in 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.

[0156] Figure 3 Electrophoresis of antibody-like molecule samples before and after cleavage by GingisKHAN protease. 7.5% PAAG, non-reducing conditions.

[0157] M - standard marker for Precision Plus Protein™ Dual Color Standard (BIO-RAD), the molecular weight of the corresponding lane is indicated in kDa in the column on the left side of the gel; 1 - 08-001 complete sample; 2 - 08-001 after protein hydrolysis; 3 - 08-002 complete sample; 4 - 08-002 after protein hydrolysis; 5 - 08-003 complete sample; 6-08-003 after protein hydrolysis.

[0158] Figure 4 Electrophoresis of antibody-like molecule samples before and after cleavage by GingisKHAN protease. 7.5% PAAG, non-reducing conditions.

[0159] M - standard marker for Precision Plus Protein™ Dual Color Standard (BIO-RAD), the molecular weight of the corresponding lane is indicated in kDa in the column on the left side of the gel; 1 - 08-004 complete sample; 2 - 08-004 after protein hydrolysis; 3 - 08-005 complete sample; 4 - 08-005 after protein hydrolysis; 5 - 08-006 complete sample; 6-08-006After protein hydrolysis.

[0160] Figure 5 Electropherograms of bispecific and monospecific antibody-like molecule samples after cleavage by GingisKHAN protease. 7.5% PAAG, non-reducing conditions.

[0161] M - standard marker for Precision Plus Protein™ Dual Color Standard (BIO-RAD), the molecular weight of the corresponding lane is indicated in kDa in the column on the left side of the gel; 1 - a bispecific antibody-like molecule comprising the variable domains of the antibodies palolizumab and ocrelizumab and a dimerization unit of the membrane-proximal domain of the CD1b protein; 2 - bispecific antibody-like molecules comprising the variable domains of the antibodies palolizumab and anti-CSF1R and a dimerization unit of the membrane proximal domain of the CD1b protein; 3 - Antibody palolizumab; 4 - monospecific antibody-like molecules comprising a dimerization unit based on the membrane-proximal domain of the CD1b protein and a variable fragment of the antibody palolizumab; 5 - A monospecific antibody comprising the variable domains of the antibody ocrelizumab. Example

[0162] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.

[0163] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended embodiments.

[0164] Materials and general methods Recombinant DNA technology Standard methods were used to manipulate DNA 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.

[0165] gene synthesis The desired gene segments were prepared from oligonucleotides prepared by chemical synthesis. Gene segments of 300-4000 bp in length were assembled by oligonucleotide annealing and ligation (including PCR amplification), flanked by unique restriction sites, and subsequently cloned via the designated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0166] DNA sequencing The DNA sequence was determined by Sanger sequencing.

[0167] DNA and protein sequence analysis and sequence data management The Ylab2 (Biocad) software package was used for sequence creation, mapping, analysis, annotation, and interpretation.

[0168] expression vector Expression plasmid variants are used to transiently express test antibodies, antibody-like proteins, and antigens in eukaryotic cells (e.g., CHO cells). In addition to the expression cassette for the target protein, the (plasmid) vector also contains: an origin of replication that allows the plasmid to replicate in E. coli, and genes that confer resistance to various antibiotics (e.g., ampicillin and kanamycin) in E. coli.

[0169] The fusion gene comprising the antibody or antibody-like molecule chain as described above is produced by PCR and / or gene synthesis and assembled by known methods and techniques through the connection of the corresponding nucleic acid segments, for example using the unique restriction sites in the corresponding vectors. The nucleic acid sequence of the subclone is verified by DNA sequencing. The necessary amount of plasmid for transient transfection is produced in Escherichia coli cell culture and isolated using known techniques.

[0170] Production and purification of recombinant antigens in mammalian cell suspension culture Recombinant proteins were produced in an established cell line derived from Chinese Hamster Ovary cells (CHO line). Suspension cultures were performed in flasks on an orbital incubator shaker using serum-free medium supplemented with 8 mM L-glutamine and 1 g / L pluronic 68. For transient expression, linear polyethyleneimine was used at a concentration of 2-2.2 × 10 6 Nine days after transfection, the culture medium was separated from the cells by filtration through a 0.22 µm filter.

[0171] The histidine-tagged protein was purified by metal chelate chromatography. The purified protein was filtered through 0.22 μm and stored at -70°C.

[0172] The purity of the resulting protein solution was assessed using SDS gel electrophoresis performed in denatured 12% PAAG in the presence of mercaptoethanol and in denatured 7.5% PAAG in the absence of mercaptoethanol.

[0173] Production of antibodies and antibody-like proteins in mammalian cell suspension culture Control antibodies and antibody-like molecules according to the present invention were produced in established cell lines derived from Chinese hamster ovary cells (CHO lines). Suspension cultures were performed in flasks on an orbital incubator shaker using serum-free medium supplemented with 8 mM L-glutamine and 1 g / L pluronic 68. For transient expression, linear polyethyleneimine (hereinafter referred to as PEI) was used at a concentration of 2-2.2 × 10 6Cells were transfected 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 measured on a ForteBio using standard methods. The clarified culture medium was passed through a Protein A affinity adsorbent column at 10-20 mg / ml of adsorbent, and the column was equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 column volumes of PBS to remove nonspecifically bound components. Bound protein was 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. The protein was then dialyzed into acetate buffer (pH 5.0), filtered (0.22 µm), transferred to a tube, and stored at -70°C.

[0174] The purity of the resulting protein solution was assessed using SDS gel electrophoresis under reducing and non-reducing conditions and using size exclusion high performance liquid chromatography (SEHPLC).

[0175] SE HPLC was performed on a TSK Gel G3000SWXL column, 7.8x300 mm, particle size: 5 μm, pore size: 250 Å, and a TSK Gel-Guard SWx1 pre-column.

[0176] Example 1. Selection of CH1 / CK domain substitutions in antibody-like molecules To ensure proper heterodimerization of the heavy and light chains in the antibody-like molecule, one of the two pairs of CH1 / CK domains is replaced with a structurally identical domain from another protein.

[0177] This results in a dimerization unit composed of: 1) the native sequence of the β2 microglobulin (β2M) of the human CD1b protein having the amino acid sequence of SEQ ID NO: 1 and 2) the α3 membrane-proximal domain of human CD1b with S12K and N59D mutations and a GSC extension at the C-terminus, having the amino acid sequence of SEQ ID NO: 2. This dimerization unit within an antibody-like molecule allows the production of functional antibody-like molecules of sufficient purity with different variable domains (as shown in the examples below), and also increases the temperature stability of the molecule and the yield of correctly assembled antibody-like molecules.

[0178] Example 2. Generation of genetic constructs for producing antibody-like molecules To generate constructs encoding light and heavy chain sequences of the first antigen-binding fragment of an antibody-like molecule that specifically binds to a first target, primers containing restriction sites are used to generate PCR products of genes containing the heavy and light chain variable domains of the antibody-like molecule. SalI / XbaI The heavy chain variable domain was cloned into the vector pSXn-HC-NR_VH1 using restriction sites. SalI / XbaI The light chain variable domain was cloned into the vector pSXn-CL-BR_VL1 using restriction sites.

[0179] To generate a structure encoding the light and heavy chain sequences of the second antigen-binding fragment of an antibody-like molecule that specifically binds to a second target, a construct comprising modified sequences encoding the heavy and light chain variable domains of an antibody-like molecule fused to the membrane proximal domain of the human CD1b protein (https: / / www.rcsb.org / structure / 5wl1) was synthesized.

[0180] Sequences were synthesized from oligonucleotides by PCR using primers containing restriction sites. SalI / XbaI The heavy chain variable domain with the modified first human CD1b sequence membrane proximal domain was cloned into the vector pSX-FC-pole-PR using restriction sites. SalI / XbaI The light chain variable domain with the membrane proximal domain of the second human CD1b sequence with or without modification was cloned into the vector pSX-HR using the restriction sites of SEQ ID NO: 1.

[0181] The above four vectors are combined in the transfection step to produce the antibody-like molecules according to the present invention.

[0182] All above plasmids were produced in desired amounts in E. coli cells and purified using Maxiprep Qiagen kit.

[0183] Example 3. Generation of Antibody-like Molecules Comprising Human CD1b Protein Dimerization Units To examine the versatility of the subject method as a platform solution for assembling antibody-like molecules using any pair of antigen-binding fragments (hereinafter referred to as light chain and heavy chain variable fragments), we selected three random pairs of light chain and heavy chain variable fragments (see Table 1) from known antibodies and used them to generate 6 antibody-like molecules. Table 1 shows the productivity results of the produced proteins. Figure 1 and 2 The results of SDS gel electrophoresis of samples produced under non-reducing and reducing conditions are shown. For all six samples under non-reducing conditions, a major band of approximately 150 kDa was present, corresponding to the full-length molecule. Table 1 also shows the sample purity results based on SE HPLC.

[0184] Table 1. Productivity and purity of antibody-like molecules produced using the modified α3 ​​domain of human CD1b protein and the dimerization unit of β2-microglobulin of human CD1b protein. Therefore, the antibody-like molecules having a dimerization unit according to the present invention having various combinations of antigen-binding fragments showed high purity and productivity.

[0185] Example 4. Determination of affinity of full-length antibody-like molecules on Forte Bio Octet RED 384 To confirm that the generated antibody-like molecules had not lost their antigen-binding ability, we performed affinity analysis on the Forte Bio OctetRED 384. For antibody-like molecules 08-001 and 08-002, we measured the affinity to the extracellular domain of human PD-1 protein (hPD1ex-H6F) and to the biotinylated peptide [NH2]CEPANPSEKNSTQYCYSIQS[CH2CH2]biotin (hereinafter referred to as CD20 peptide) containing a fragment of the human CD20 sequence in its amino acid sequence; for antibody-like molecules 08-003 and 08-004, we measured the affinity to the extracellular domain of human PD-1 protein (hPD1ex-H6F) and to the extracellular domain of human CSF1R protein (hCSF1R_His); for antibody-like molecules 08-005 and 08-006, we measured the affinity to the extracellular domain of human CSF1R protein (hCSF1R_His) and to the CD20 peptide.

[0186] As the hCSF1R antigen, we used the amino acid sequence 20-512 (hereinafter referred to as AA) of the human CSF1R protein (macrophage colony-stimulating factor 1 receptor (Homo sapiens), UNIPROT ID P07333) with a C-terminal His-tag and FLAG tag, with a molecular weight of 57.4 kDa. As the hPD-1ex-H6F antigen, we used the amino acid sequence 21-170 of the human PD-1 protein (programmed cell death protein 1 (Homo sapiens), UNIPROT ID Q15116) with a C-terminal His-tag and FLAG tag, with a molecular weight of 20.6 kDa.

[0187] The genetic sequence encoding the antigen was synthesized de novo, cloned into an expression vector, produced in CHO cells and purified using affinity chromatography as described in the general examples.

[0188] Experiments were performed using kinetic buffer (hereafter referred to as 1xKB) containing 0.1% Tween 20 and 0.1% BSA (bovine serum albumin) at a pH of 7.4. Prior to measurement, the ProA sensor was regenerated with a solution of 50 mM glycine and hydrochloric acid (pH 1.8) (5 seconds in regeneration buffer, 5 seconds in 1xKB, repeated three times).

[0189] For the hPD1ex-H6F and hCSF1R_His antigens, a Protein A (ProA) biosensor (ForteBio) was immersed in a solution containing the antibody-like molecule at a concentration of 10 μg / ml for 300 seconds to immobilize it. A baseline was recorded in 1xKB for 120 seconds. The sensor loaded with the antibody-like molecule was then immersed in a well containing a solution of the target antigen (analyte) in kinetic buffer for 300 seconds. Measurements were performed on solutions containing the hPD1ex-H6F analyte at a concentration of 10 μg / ml (485.4 nM) and the hCSF1R_His analyte at a concentration of 10 μg / ml (174.2 nM). Complex dissociation was then monitored in 1xKB for 600 seconds.

[0190] The reference sensor underwent all the steps as the sensor used to record the analyte sensorgram, except for the association step, in which the sensor was immersed in a 1xKB solution without analyte (the reference sensor signal was measured in parallel with the recording of the main sensorgram). During the processing of the sensorgram, the reference signal was subtracted from the signal received on the sensor interacting with the analyte.

[0191] To examine nonspecific interactions between analytes and sensors, we used sensors not loaded with antibody-like molecules (in the loading step, the sensor was immersed in 1xKB solution; all other steps were the same as those for the sensor loaded with antibody-like molecules).

[0192] In the case of biotinylated CD20 peptide, the peptide was immobilized at a concentration of 2.5 μg / ml on the surface of a SAX sensor (High Precision Streptavidin (SAX) biosensor, ForteBio) for 300 seconds. A baseline was recorded in a 1xKB solution for 120 seconds. The peptide-loaded sensor was then immersed in a well containing a solution of an antibody-like molecule (analyte) in 1xKB for 300 seconds. Measurements were performed on solutions containing the analyte (antibody-like molecule in the case of CD20 peptide) at concentrations of 300 μg / ml and 75 μg / ml. Complex dissociation was recorded in 1xKB for 60 seconds.

[0193] All measurements were performed at 30 °C with orbital mixing at 1000 rpm.

[0194] To obtain values ​​for kinetic constants (k is the association / association rate constant, k is the dissociation rate constant, and K is the equilibrium dissociation constant or affinity constant), the sensorgrams were processed according to a 1:1 interaction model using Global Fit (a set of k, k, and K constants was selected to analyze several sensorgrams at different concentrations) in ForteBio Octet Data Analysis 9.0 software. The results are shown in Table 2.

[0195] Table 2. Kinetic constants kon, kdis, and KD for the interactions between antibody-like molecules 08-001 to 08-006 and target antigens. Table 3. Validation results showing nonspecific interactions between analytes and unloaded sensors. No nonspecific interactions between analytes and unloaded sensors were detected.

[0196] Table 3. Verification results of nonspecific interactions between analytes and unloaded sensors From the results provided, it can be seen that all antibody-like molecules 08-001 - 08-006 showed specific binding to the target antigen; furthermore, the KD values ​​were of the same or similar order of magnitude, regardless of the location of the corresponding antigen-binding fragment (as part of a Fab fragment or as part of a Fab-like fragment containing a modified α3 ​​domain of β2 microglobulin and CD1b protein instead of the CK and CH1 domains).

[0197] Example 5. Analysis of simultaneous binding of antibody-like molecules to two antigens according to the present invention. The antibody-like molecules 08-003 and 08-004 were analyzed for simultaneous binding to two different target antigens (hPD1ex-H6F and hCSF1R-His) on a Forte Bio Octet RED 384. The experiments were performed on an AR2G sensor (Amine Reactive Second-Generation (AR2G) biosensor, ForteBio). The experimental steps are shown in Table 4.

[0198] Table 4. Experimental steps to verify the simultaneous binding of the antibody-like molecule according to the present invention to two different antigens The sensor was activated for 300 seconds in an aqueous solution containing 20 mM EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 10 mM sNHS (N-hydroxysulfosuccinimide). Antigen (hPD1ex-H6F) was loaded onto the biosensor surface in 10 mM sodium acetate buffer (pH 5.0) for 300 seconds. The loaded protein concentration was 10 μg / ml. Unreacted active sites on the sensor surface were quenched in 1 M ethanolamine aqueous solution (pH 8.5) for 300 seconds. The baseline of the fifth step and all subsequent steps were performed in kinetic buffer (1xKB) with a 0.1% volume fraction of Tween 20 and a 0.1% mass fraction of BSA, pH 7.4. After recording the baseline (step 5), antibody-like molecules were loaded onto the sensor at a concentration of 10 μg / ml (step 6). A third baseline was then recorded (step 7). This step showed the absence of a rapid signal decay, indicating a specific interaction between the loaded antibody-like molecule and the hPD1ex-H6F previously immobilized on the sensor. In the next association step (step 8), the sensor containing immobilized hPD1ex-H6F and bound antibody-like molecule was immersed in a solution of hCSF1R_His antigen at a concentration of 10 μg / ml. The signal amplification in this step is due to the presence of the FAB fragment of the hCSF1R_His antigen in the antibody-like molecule loaded on the sensor.

[0199] The sensorgrams were analyzed using ForteBio Octet Data Analysis 9.0 software. The main results are shown in Table 5. According to the results, samples 08-003 and 08-004 showed simultaneous binding to hPD1ex-H6F and hCSF1R_His antigens.

[0200] Table 5. Results of experiments determining the simultaneous interaction of antibody-like molecules 08-003 and 08-004 with two different antigens (hPD1ex-H6F and hCSF1R_His). According to the results, the antibody-like molecules 08-003 and 08-004 showed simultaneous binding to hPD1ex-H6F and hCSF1R_His antigens.

[0201] Example 6. Analysis of the Molecular Weight of Antibody-Like Molecules According to the Present Invention by Reverse Phase Ultra-High Performance Liquid Chromatography (RP UHPLC) with Mass Spectrometry Detection To confirm the correct assembly of the molecules, the molecular weight of the full-length antibody-like molecules according to the invention was analyzed. This method made it possible to identify full-length antibody-like molecules according to the invention composed of four different chains and to distinguish them from byproducts formed by another set of chains. These data, as well as those from Examples 4 and 5, demonstrate that the technical solution based on replacing the constant domain CH1-CK pair with a pair consisting of the β2 microglobulin and modified α3 ​​domains of the human CD1b protein ensures the correct assembly of the antibody-like molecules according to the invention.

[0202] Analysis was performed using an Agilent 1290 Infinity II UPLC coupled to an Agilent 6530 Q-Tof HPLC-MS / MS system on a BioResolve Polyphenyl RP column (2.1 x 50 mm, 2.7 μm particle size; a BioResolve Polyphenyl RP pre-column, 2.1 x 5 mm, 2.7 μm particle size, was also used). Prior to analysis, all molecules were treated with PNGase F (Promega) to remove N-glycans. To this end, the enzyme was diluted with water to a concentration of 1 unit activity per μl. A 50 μg sample of protein was mixed with buffer solution, and the PNGase F solution was added at a ratio of 1 unit:50 μg enzyme:protein. The mixture was incubated in a thermostat at 37.0 ± 0.1°C for 18 hours.

[0203] For analysis, 5 μg of test solution was selected based on the measured protein concentration and the concentration was adjusted to 0.2 mg / ml using mobile phase A. The sample input volume was 5 μl.

[0204] Prior to analysis, the chromatographic system was equilibrated with mobile phases at an initial ratio of A:95%, B:5% (Phase A contained 0.1% formic acid in water, Phase B contained 0.1% formic acid solution, 30% acetonitrile in isopropanol) for at least 30 minutes until a stable pressure was achieved. The mass spectrometer was calibrated using calibration standards according to the manufacturer's guidelines.

[0205] The samples were separated with a concentration gradient of mobile phase B (5%-30% in 2-3 minutes after sample introduction, then 30%-35% in 3-8 minutes, and then 35-95% in 8-9 minutes) at a flow rate of 0.45 ml / min at a column temperature of (60 ± 1)°C; chromatograms were obtained at a wavelength of 280 nm.

[0206] The data were processed using Protein Metrics.

[0207] The results of mass analysis of antibody-like molecules according to the present invention are shown in Table 6. In this table, each antibody-like molecule is provided with the percentage of the mass corresponding to a correctly assembled antibody-like molecule consisting of four different chains relative to all masses measured by the instrument.

[0208] Table 6. Content of correctly and incorrectly assembled antibody-like molecules in samples treated with PNGase F The analysis results allow to conclude that the mass of full-sized antibody-like molecules composed of four different chains is the predominant one in all samples.

[0209] Example 7. Confirmation of the correct assembly of antibody-like molecules according to the present invention using proteolysis and mass analysis of the resulting fragments To directly verify the correct pairing of the light and heavy chains, the antibody-like molecules according to the present invention were cleaved by GingisKHAN protease (Genovis), whose recognition site is located in the hinge region of the antibody-like molecule (...KSCDK / THTCPPCP...). This proteolysis results in the breakdown of the full-length antibody-like molecule into an Fc fragment, a Fab fragment, and a Fab-like fragment containing a β2 microglobulin and a modified α3 ​​domain of human CD1b protein replacing the CK and CH1 domains. Following proteolysis, the fragments of the antibody-like molecule were analyzed using vertical electrophoresis under non-reducing conditions; the resulting fragments were also subjected to mass spectrometry analysis.

[0210] The proteolysis reaction mixture contained 40 μg of antibody-like molecules and 40 units of GingisKHAN enzyme (ratio of 1 enzyme unit: 1 μg protein) in a 60 μl volume of a buffer consisting of 100 mM Tris-HCl (pH 8.0) and 1 mM cysteine. The reaction was carried out in an incubator at (37.0 ± 0.1)°C for 1 hour and stopped by adding iodoacetamide to a concentration of 10 mM. For electrophoresis, a buffer containing SDS (up to a concentration of 1% SDS) was added to the resulting sample, and SDS gel electrophoresis was performed under non-reducing conditions.

[0211] Figure 3-5 The results of SDS gel electrophoresis are shown. After the antibody-like molecules (08-001-08-006) were treated with GingisKHAN protease ( Figure 3 and 4 ) formed three major fragments, which were observed in the 40-50 kDa region of the electrophoretogram and showed different mobilities in PAAG. A monospecific molecule ( comprising a dimerization unit of the membrane proximal domain of the human CD1b protein and comprising a variable fragment of the antibody palolizumab) was also treated with GingisKHAN protease. Figure 5, lane 4), and monospecific antibodies in IgG1 format comprising the variable fragments of the antibody palolizumab and the antibody ocrelizumab ( Figure 5 , lanes 3, 5). Comparison of the electrophoretic mobility of fragments formed during proteolysis of monospecific and bispecific antibody-like molecules revealed that the fragment with the lowest mobility corresponds to the Fc fragment, the fragment with intermediate mobility corresponds to the Fab fragment, and the fragment with the highest mobility corresponds to a Fab-like fragment containing β2 microglobulin and a modified α3 ​​domain of the CD1b protein, replacing the CK and CH1 domains. In the case of antibody-like molecules 08-003 - 08-006, the electropherograms revealed the formation of another fragment due to nonspecific cleavage of the variable domain of the anti-CSF1R fragment. To determine the exact masses of the fragments formed by proteolysis, mass spectrometry analysis was performed.

[0212] Prior to mass spectrometry analysis, samples were transferred to 50 mM ammonium bicarbonate (pH (7.6 ± 0.2)) on a Zeba (MWCO 7 kDa) column immediately after the addition of iodoacetamide according to the manufacturer's instructions; PNGase F was then added to the sample at a ratio of 1 enzyme unit:50 μg protein and the sample was incubated at (37.0 ± 0.1)°C for 18 hours.

[0213] Mass analysis of the resulting fragments was performed as described in Example 6 with the following changes: to achieve better peak resolution, the samples were separated using a concentration gradient of mobile phase B (5% to 55% from 2 to 20 minutes after sample introduction, then 55% to 95% from 20 to 21 minutes) at a column temperature of (60 ± 1) °C and a flow rate of 0.4 ml / min; phase A consisted of 0.1% formic acid, 0.02% trifluoroacetic acid in water, and phase B consisted of 0.1% formic acid solution, 30% acetonitrile in isopropanol.

[0214] The RP UHPLC and mass spectrometry detection results are shown in Table 8.

[0215] Table 8. Fragment contents in samples treated with GingisKHAN and PNGase F The results showed that all antibody-like molecules according to the present invention were decomposed after proteolysis into fragments with masses corresponding to the Fc fragment, the Fab fragment and the Fab-like fragment containing the β2 microglobulin of the human CD1b protein replacing the CK and CH1 domains and the modified α3 ​​domain, indicating that the technical solution based on replacing the constant domain CH1-CK pair with a pair consisting of the β2 microglobulin of the human CD1b protein and the modified α3 ​​domain provides the correct assembly of the 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 comprises: 1) a first antigen-binding fragment that specifically binds to a first target and comprises: a) a light chain of the first antigen-binding fragment, wherein the light chain comprises a light chain variable domain and a light chain constant domain; and b) a heavy chain of a first antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain of an antibody and a heavy chain constant domain of an Fc fragment monomer comprising a first (CH1) heavy chain constant domain and a second (CH2) and a third (CH3) heavy chain constant domain; and 2) a second antigen-binding fragment that specifically binds to a second target and comprises: (i) a) a light chain of a second antigen-binding fragment, the light chain comprising a light chain variable domain and a constant domain of β2 microglobulin (β2M), which is a human CD1b (cluster of differentiation 1) protein having the amino acid sequence of SEQ ID NO: 1; and b) a heavy chain of a second antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain, a constant domain that 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 second (CH2) and third (CH3) heavy chain constant domains; or (ii) a) a light chain of a second antigen-binding fragment, wherein the light chain comprises a light chain variable domain and a constant domain that is a human CD1b protein α3 membrane proximal domain having the amino acid sequence of SEQ ID NO: 2; and b) a heavy chain of a second antigen-binding fragment, the heavy chain of which comprises a heavy chain variable domain, a constant domain of β2 microglobulin (β2M) which is a 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; 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 stabilized by a disulfide bond therebetween.

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

3. The antibody-like molecule according to claim 2, wherein a) the CH3 domain of the one heavy chain is modified such that, on the surface of the one heavy chain CH3 domain contacting the surface of the other heavy chain CH3 domain in the antibody-like molecule, the 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 one heavy chain CH3 domain that can fit into the hole on the surface of the other heavy chain CH3 domain, and b) the CH3 domain of the other heavy chain is modified such that on the surface of the second heavy chain CH3 domain contacting the surface of the first heavy chain CH3 domain in the antibody-like molecule, the amino acid residue is substituted by an amino acid residue with a smaller side chain volume, resulting in the formation of a hole on the surface of the second heavy chain CH3 domain that is capable of fitting into the knob on the interface of the first heavy chain CH3 domain; wherein the amino acid residue with a larger side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W), and wherein the amino acid residue with a smaller side chain volume is selected from alanine (A), serine (S), threonine (T), and valine (V). The antibody-like molecule according to claim 1 , wherein the first light chain constant domain of the antibody-like molecule is selected from CK or CL.

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 into the corresponding position of each CH3 domain so that a disulfide bond can be formed between the two CH3 domains.

6. The antibody-like molecule according to any one of claims 2-3, wherein 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.

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

8. antibody-like molecule according to claim 6, the CH3 domain of wherein said one heavy chain has Y349C / T366S / L368A / Y407 amino acid substitution according to the EU numbering scheme for antibody amino acids, and the CH3 domain of said other heavy chain has S354C / T366W amino acid substitution according to the EU numbering scheme for antibody amino acids. 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 human IgG1, IgG2 or IgG4.

11. The antibody-like molecule according to claim 1, wherein the Fc fragment monomer comprises a substitution resulting in no 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 comprises L234A and L235A substitutions according to the EU numbering scheme for antibody amino acids.

13. The antibody-like molecule of claim 1, wherein the Fc fragment monomer comprises a substitution that results in an elongation of the antibody-like molecule.

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

15. The antibody-like molecule of claim 1, wherein the Fc fragment monomer comprises 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 comprises an E345R substitution according to the EU numbering scheme for antibody amino acids.

17. The antibody-like molecule of any one of claims 1-16, wherein the first and second targets are each independently selected from the group consisting of CD20, BCMA, PD-1, PD-L1, CD47, GD2, AXL, TGFβ, CSF1R, coagulation factor 9 (FIX), coagulation factor 10 (FX), TNFα, 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 according to any one of claims 1 to 16, wherein the antibody-like molecule is a bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, heptaspecific, octaspecific or nonaspecific antibody-like molecule.

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

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

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

23. An expression vector comprising the nucleic acid according to any one of claims 21-22.

24. A method for producing a host cell to produce an antibody-like molecule according to any one of claims 1 to 20, comprising transforming the cell with a 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 a nucleic acid according to claims 21 to 22.

26. A method for producing an antibody-like molecule according to claims 1-20, wherein the method comprises the following steps: a) Transform the host cell with - an expression vector comprising a nucleic acid molecule encoding the light chain and the heavy chain of the first antigen-binding fragment of the antibody-like molecule, - an expression vector comprising a nucleic acid molecule encoding the light chain and the heavy chain of the second antigen-binding fragment of the antibody-like molecule, b) culturing the host cell under conditions suitable for synthesis of the antibody-like molecule; and c) isolating the antibody-like molecule from the cell culture.

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