RU2818312C9 - Bispecific antibody or antigen-binding fragment thereof and method for production thereof - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to a protein complex characterized by high index of heterodimer formation, and to a method for its preparation.

EFFECT: invention enables to obtain protein complexes with improved efficiency of heterodimer formation for use in various fields, such as prevention or treatment of diseases and diagnosis of diseases.

25 cl, 10 dwg, 12 tbl, 8 ex

Description

TECHNICAL FIELD

This application claims priority to Korean Patent Application No. 10-2020-0066030, filed June 1, 2020, the disclosure of which is incorporated herein in its entirety.

The present invention relates to a bispecific antibody characterized by a high heterodimer formation rate or an antigen binding fragment thereof and a method for its production.

BACKGROUND OF THE ART

Natural human immunoglobulin (Ig) consists of two identical heavy chains and two light chains linked together. In addition, immunoglobulins can be functionally divided into an antigen-binding fragment (Fab) region, which forms antigen-antibody binding, and a crystallizable fragment (Fc) region, in which the two heavy chains form a dimer. These natural immunoglobulins, i.e. Monoclonal antibodies (mAbs) typically target the same binding site (epitope) of the same antigen because the two Fab regions are identical to each other and they have bivalent and monospecific binding properties.

Immunoglobulins are widely used as a therapeutic agent for various diseases due to the target-specific antigen-binding function of the Fab region as well as the function of the Fc region to induce immune cells and increase half-life in vivo. However, recently, for intractable diseases such as cancer or autoimmune diseases, synergistic treatment using two drugs acting on different targets has shown greater therapeutic effect than using a single drug, so various bispecific antibodies are being developed that can act on two target substances.

Among the bispecific antibodies developed in this way, bispecific antibodies of the scFv-scFv type, in which the variable region fragments of different antibodies are linked by a polypeptide chain, have the disadvantage that they are not able to remain in the body for a long time and maintain effectiveness due to their low molecular weight and low stability. In addition, bispecific antibodies of the scFv-Fab-Fc or Fab-Fc-scFv type, in which the end of a natural immunoglobulin polypeptide is linked to a fragment of the variable region of another antibody, are characterized by the disadvantages associated with the possibility of forming an aggregate due to low structural stability, while they exhibit immunogenicity in vivo. Therefore, to solve these problems, it is necessary to develop bispecific antibodies containing different variable regions that maximally replicate the form of natural immunoglobulin.

To form this type of bispecific antibody, a heterodimer must form between the Fc regions of two different Fab-Fc complexes. Since amino acids at positions where CH3 domains interact with each other play an important role in the formation of the Fc heterodimer, Fc heterodimer technology is being developed in such a way that these amino acids are replaced by other amino acids (US Patent No. 5731168 A (1998.03.24), Korean Patent No. 10-2098919 (2020.04.02)).

However, the existing Fc heterodimer technology developed in this manner also has the limitation that it cannot produce all Fc structures in the form of 100% heterodimers, that is, a mixture of heterodimers and homodimers is formed.

Therefore, there is a need to develop a protein complex with improved heterodimer formation efficiency, a bispecific antibody containing it or an antigen-binding fragment thereof, receptors and receptor-binding agonists, antagonists, ligands, decoy receptor-based biconjugates, and the like.

DESCRIPTION OF IMPLEMENTATION OPTIONS TECHNICAL PROBLEM

In one aspect, a protein complex is provided that has a high rate of heterodimer formation.

In another aspect, a method is provided for producing a protein complex having a high rate of heterodimer formation.

In yet another aspect, a pharmaceutical composition for the prevention or treatment of a disease is provided, comprising a protein complex characterized by a high rate of heterodimer formation.

In yet another aspect, a method is provided for preventing or treating a disease using a protein complex having a high rate of heterodimer formation.

In yet another aspect, uses of a protein complex having a high rate of heterodimer formation are provided for the preparation of a prophylactic or therapeutic agent for the prevention or treatment of a disease.

THE SOLUTION OF THE PROBLEM

In one aspect, there is a protein complex comprising a first polypeptide comprising a first antibody constant region CH3 and a second polypeptide comprising a second antibody constant region CH3, wherein the first polypeptide and the second polypeptide form a heterodimer.

In one embodiment, the first antibody constant region CH3 includes tryptophan (W) at position 366, and the second antibody constant region CH3 includes series (S) at position 366, alanine (A) at position 368, and valine (V) at position 407; and at least one of the first CH3 antibody constant region and the second CH3 antibody constant region may include at least one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), histidine (H), glycine (G) , valine (V), methionine (M) and alanine (A), at one or more positions selected from the group consisting of positions 351 and 394.

The protein complex includes a first polypeptide comprising a first CH3 constant region of an antibody, and a second polypeptide comprising a second CH3 constant region of an antibody, wherein the first polypeptide and the second polypeptide form a heterodimer.

The term "antibody" is used interchangeably with the term "immunoglobulin (Ig)". A complete antibody is characterized by a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain via a disulfide bond (SS bond). There are two types of light chains: λ and κ, each consisting of approximately 211-217 amino acids. Each human antibody has only one type of light chain. The light chain consists of a continuously connected constant region and a variable region. There are five types of heavy chains (γ, δ, α, μ, ε), with the heavy chain determining the type of antibody, α and γ consist of 450 amino acids, and μ and ε consist of 550 amino acids. A heavy chain has two regions: a variable region and a constant region. The variable region refers to the region to which an antigen binds in an antibody. The variable region may include a complementarity determining region (CDR) conferring antigen binding specificity.

An antibody may include an antigen-binding fragment (Fab) region that binds to an antigen and a crystallizable fragment (Fc) region that binds to a cell surface receptor. When digested with papain, the complete antibody can be split into two Fab regions and one Fc region. The Fab region may be a region in which a polypeptide including a heavy chain variable region (VH) domain and a heavy chain constant region domain 1 (CH1), and a polypeptide including a light chain variable region (VL) domain and a light chain constant region domain ( CL), linked by a disulfide bond. The Fc region may be a region in which two polypeptides comprising heavy chain constant region domain 2 (CH2) and heavy chain constant region domain 3 (CH3) are linked. The Fc region may form a hinge region.

The antibody constant region CH3 refers to domain 3 of the antibody heavy chain constant region.

The first polypeptide and the second polypeptide may form an Fc region of the antibody.

A heterodimer refers to a combination of two polypeptides characterized by different sequences, numbers, or types of amino acid residues. The protein complex may be a bispecific antibody or an antigen-binding fragment thereof formed by combining two polypeptides that specifically bind to targets different from each other.

The protein complex may be a bispecific antibody or antigen-binding fragment thereof, a receptor-agonist conjugate, a receptor-antagonist conjugate, a receptor-ligand conjugate, or a ligand-decoy receptor conjugate. In addition, the protein complex may include any one selected from the group consisting of an antigen binding fragment (Fab), a single chain variable fragment (scFv), a membrane receptor extracellular domain, an agonist, an antagonist, a ligand, a decoy receptor, a cytokine, a coagulation factor, and an affinity tag .

The antibody may be, for example, IgA, IgD, IgE, IgG or IgM. The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may be an animal-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.

The term "antigen-binding fragment" refers to a fragment of the structure of a complete immunoglobulin and refers to a portion of a polypeptide including a portion capable of binding to an antigen. For example, the antigen binding fragment may be scFv, (scFv) ₂ , Fv, Fab, Fab', Fv F(ab') ₂ or a combination thereof.

The term "bispecific" refers to the specific recognition of target proteins that are different from each other. A bispecific antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof having two antigen-binding sites that recognize different target antigens. A bispecific antibody or antigen-binding fragment thereof may also be referred to as a bispecific antibody (BsAb).

The bispecific antibody or antigen binding fragment thereof may be a knob-to-trench (kih) IgG, scFv-Fc, scFv2-Fc, TrioMab, IgG-like antibody, CrossMab, 2:1 CrossMab, 2:2 CrossMab, DuoBody, DVD-Ig (Dual Variable Domain Immunoglobulin), scFv-IgG, IgG-IgG, Fab-scFv-Fc, ADPTIR, BiTE (Bispecific T Cell Recruiter)-Fc, DART (Dual Affinity Retargeting Antibody) -Fc, quadrivalent DART-Fc, LP-DART, CODV (Cross-Double Variable Domain Antibody)-Ig, CODV-Fab-TL, HLE (Extended Half Life) -Bite, Tandem V _HH (Heavy Chain Single Variable Domain) )-Fc or a combination thereof.

The term "receptor" refers to a substance that receives or transmits a signal that can be transmitted to a biological system. The receptor may be a protein receptor. The receptor can bind to an agonist, antagonist, ligand, or cytokine. An agonist may be a substance that binds to a receptor and activates the receptor, initiating a biological response. An antagonist may be a substance that binds to a receptor and inhibits the receptor, suppressing the biological response. A ligand may be a substance that binds to a receptor. The ligand can bind to the decoy receptor. A decoy receptor refers to a receptor that specifically binds to a ligand, thereby inhibiting signal transmission by the actual receptor. A cytokine refers to a small protein that acts on cellular signal transduction, regulation and maintenance of inflammatory processes.

The protein complex can be modified. For example, the protein complex may be modified by conjugation or binding, glycosylation, tagging, or a combination thereof. The antibody may be conjugated to other drugs, such as anti-cancer drugs. For example, the protein complex may be combined with horseradish peroxidase (HRP), alkaline phosphatase, hapten, biotin, streptavidin, fluorescent material, radioactive material, quantum dots, polyethylene glycol (PEG), a histidine tag, or a combination thereof. The fluorescent substance may be Alexa Fluor®532, Alexa Fluor®546, Alexa Fluor®568, Alexa Fluor®680, Alexa Fluor®750, Alexa Fluor®790, or Alexa Fluor®350.

The amino acid position corresponds to Kabat's EU index (EU index as described in 'Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)') . The amino acid position in the CH3 domain and the corresponding amino acid type are based on human IgG1.

In a protein complex, the first CH3 of the antibody constant region may include tryptophan (W) at position 366. In the first CH3 of the antibody constant region, position 366 may be a position (T366W) in which threonine (T) is replaced by tryptophan (W). The second CH3 of the antibody constant region may include series (S) at position 366, alanine (A) at position 368, and valine (V) at position 407. In the second CH3 of the antibody constant region, position 366 may be the position (T366S) at which threonine ( T) is replaced by serine (S). In the second CH3 constant region of the antibody, position 368 may be a position (L368A) in which leucine (L) is replaced by alanine (A). In the second CH3 constant region of the antibody, position 407 may be a position (Y407V) in which tyrosine (Y) is replaced by valine (V).

In the protein complex, at least one of the first CH3 antibody constant region and the second CH3 antibody constant region may include at least one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), histidine (H), glycine ( G), valine (V), methionine (M), alanine (A), isoleucine (I) and serine (S), at one or more positions selected from the group consisting of positions 351 and 394.

For example, the first CH3 of the antibody constant region may include at least one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), histidine (H), glycine (G), valine (V), methionine (M) , alanine (A) and isoleucine (I), at one or more positions selected from the group consisting of positions 351 and 394. In addition, the second CH3 of the antibody constant region may include at least one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), histidine (H), glycine (G), valine (V), methionine (M), alanine (A) and serine (S), at one or more positions selected from the group, consisting of provisions 351 and 394.

In one embodiment, the first CH3 of the antibody constant region may include tryptophan (W) at position 366 and phenylalanine (F), histidine (H), or tryptophan (W) at position 394. Position 394 may be the position at which threonine (T) replaced by phenylalanine (F), histidine (H), or tryptophan (W) (T394F, T394H, or T394W).

In another embodiment, the first CH3 of the antibody constant region may include tryptophan (W) at position 366 and may include tryptophan (W), valine (V), alanine (A), or phenylalanine (F) at position 351. Position 351 may be position , in which leucine (L) is replaced by tryptophan (W), valine (V), alanine (A), or phenylalanine (F) (L351W, L351V, L351A, or L351F).

In another embodiment, the second CH3 constant region of the antibody may include series (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and alanine (A), glycine (G), valine (V), methionine (M) or phenylalanine (F) at position 351. Position 351 may be a position in which leucine (L) is replaced by alanine (A), glycine (G), valine (V), methionine (M) or phenylalanine (F ) (L351A, L351G, L351V, L351M or L351F).

The first CH3 of the antibody constant region may include one selected from the group consisting of:

tryptophan (W) at position 366;

tryptophan (W) at position 366 and histidine (H) at position 394;

tryptophan (W) at position 366 and phenylalanine (F) at position 394;

tryptophan (W) at position 366 and tryptophan (W) at position 394;

tryptophan (W) at position 366 and phenylalanine (F) at position 351;

tryptophan (W) at position 366 and tryptophan (W) at position 351;

tryptophan (W) at position 366 and valine (V) at position 351;

tryptophan (W) at position 366 and alanine (A) at position 351; And

tryptophan (W) at position 366, histidine at position 394 (H), and phenylalanine at position 351 (F).

The second CH3 constant region of the antibody may include one selected from the group consisting of:

serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407;

serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and alanine (A) at position 351;

serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and glycine (G) at position 351;

serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and valine (V) at position 351;

serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and methionine (M) at position 351; And

serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and phenylalanine (F) at position 351.

The first CH3 of the antibody constant region may include tryptophan (W) at position 366 and phenylalanine (F), histidine (H), or tryptophan (W) at position 394; and the second CH3 constant region of the antibody may include ser (S) at position 366, alanine (A) at position 368, and valine (V) at position 407.

The first CH3 of the antibody constant region may include tryptophan (W) at position 366 and phenylalanine (F) at position 351; and the second CH3 of the antibody constant region may include series (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and alanine (A) at position 351. The first CH3 of the antibody constant region may further include histidine ( H) in position 394.

The first CH3 of the antibody constant region may include tryptophan (W) at position 366; and the second CH3 of the antibody constant region may include series (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and glycine (G) at position 351. The first CH3 of the antibody constant region may further include phenylalanine ( F) or tryptophan (W) at position 351.

The first CH3 of the antibody constant region may include tryptophan (W) at position 366; and the second CH3 constant region of the antibody may include series (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and alanine (A), phenylalanine (F), valine (V), or methionine (M) ) at position 351. The first CH3 of the antibody constant region may further include a valine (V) or alanine (A) at position 351. For example, the first CH3 of the antibody constant region may include tryptophan (W) at position 366 and valine (V) at position 351 ; and the second CH3 of the antibody constant region may include series (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and alanine (A) at position 351. In addition, the first CH3 of the antibody constant region may include tryptophan (W) at position 366 and alanine (A) at position 351; and the second CH3 constant region of the antibody may include series (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and phenylalanine (F) at position 351.

The protein complex, in one aspect, may be a reverse sequence substituted Fc variant. As used herein, the term “reverse sequence variant” refers to a structure in which the left and right heavy chain constant regions of a protein complex are symmetrical about the Y axis of the coordinate plane. For example, a reverse sequence substitution variant has the same specific position and type of amino acid substitution in the protein complex of the embodiment, but the first antibody constant region CH3 and the second antibody constant region CH3 may have a structure symmetrical about the Y axis on the coordinate plane. Because the reverse sequence substitution variant forms a heterodimer with an index similar to that before the specific amino acid substitution, it is possible to form an Fc variant with high efficiency. Specific details regarding the protein complex are described above.

In one embodiment, the first CH3 of the antibody constant region may include ser (S) at position 366, alanine (A) at position 368, and valine (V) at position 407; and the second CH3 of the antibody constant region may include tryptophan (W) at position 366; and at least one of the first CH3 antibody constant region and the second CH3 antibody constant region may include at least one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), histidine (H), glycine (G) , valine (V), methionine (M), and alanine (A), at one or more positions selected from the group consisting of positions 351 and 394. Specific details regarding the first CH3 antibody constant region and the second CH3 antibody constant region are described higher.

In addition, the first CH3 of the antibody constant region may include series (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and glycine (G) at position 351; and the second CH3 of the antibody constant region may include tryptophan (W) at position 366. The second CH3 of the antibody constant region may further include phenylalanine (F) or tryptophan (W) at position 351.

In one embodiment, by comparing the heterodimer formation rates of a wild-type IgG1 bispecific antibody and a Genentech bispecific antibody generated by the knob-in-hole (KiH) approach with the formation rate of Fc variants selected after amino acid substitutions at certain positions in the CH3 constant region of the antibody, it was confirmed that the heterodimer formation rate of the Fc variants was markedly higher, while the thermodynamic stability was also excellent. In addition, the rate of heterodimer formation was also confirmed to be markedly higher in the reverse sequence substituted Fc variants compared to wild-type IgG1 and the Genentech bispecific antibody generated by the knob-in-hollow (KiH) approach. Thus, the protein complex, in one aspect, has improved heterodimer formation efficiency, and therefore the protein complex can be used as a bispecific antibody comprising itself or its antigen-binding moiety, receptor, and receptor-binding agonist, antagonist, ligand, cytokine, biconjugate based on decoy receptor, etc.

In another aspect, a method for producing a protein complex is provided, comprising transforming one or more cells with one or more expression vectors encoding a first polypeptide of one aspect, a second polypeptide of one aspect, or a combination thereof; and causing expression of the first polypeptide, the second polypeptide, or a combination thereof.

Specific details regarding the first polypeptide, the second polypeptide and the protein complex are described above.

"Expression vector" refers to an expression vector capable of expressing a target protein in appropriate host cells, and it refers to a vector comprising key regulatory elements operably linked such that the inserted nucleic acid sequence can be expressed. "Operably linked" means that the regulatory sequence for controlling the expression of the nucleic acid and the nucleic acid encoding the target protein are operably linked to perform a common function. The expression vector may include a polynucleotide encoding a first polypeptide, a second polypeptide, or a combination thereof. The expression vector may include a regulatory region necessary for gene expression, such as an enhancer, promoter, poly(A) sequence, and the like.

The cells may be cancer cells. The cells may be in vitro cells. The cells may be bacteria, yeast, plant cells or mammalian cells. The bacteria may be E. coli. Mammalian cells refer to cells obtained from mice, rats, rabbits, dogs, cats, sheep, cows, horses, monkeys, chimpanzees or humans. The cells may be a cell line. The cells may be selected, for example, from the group consisting of Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, baby hamster kidney (BHK) cells, NS0 cells, PER.C6 cells, HeLa cells, canine kidney cells Madin-Darby (MDCK), SP2/0 mouse myeloma cells, COS-7 cells and YB2/0 rat myeloma cells. The CHO cells may be CHO DG44, CHO-K1, CHO-S, GS-CHO, or CHO DUKX (DXB11) cells. The HEK cells may be HEK 293 cells.

"Transformation" refers to a method of inserting a specific nucleic acid fragment into the genome of a cell such that the inserted nucleic acid is expressed.

Expression vectors encoding a first polypeptide and expression vectors encoding a second polypeptide can be co-transfected into cells, or expression vectors encoding a first polypeptide and expression vectors encoding a second polypeptide can be transformed into at least two cell types, with one vector for each cell type.

Cells can be cultured in cell culture medium. Cell culture medium refers to a solution containing nutrients necessary for culturing cells. The medium includes commercial or prepared medium used for cell culture. The cell culture medium may contain an antibiotic. The cell culture medium may include G418 (geneticin), puromycin, blasticidin, zeocin, or a combination thereof. The cell culture medium may include a chemically defined medium.

The cell may be cultured under conditions that allow cell survival or proliferation. The conditions that ensure cell survival or proliferation may vary depending on the cell type. Cells can be cultured at a temperature of about 25°C to about 42°C, from about 25°C to about 40°C, from about 30°C to about 40°C, from about 30°C to about 37°C, or at approximately 37°C. Cells can be cultured in the presence of air containing from about 1% CO ₂ to about 10% CO ₂ or from about 5% CO ₂ to about 10% CO ₂ . Cells can be cultured in a pH of about pH 6 to about pH 8, about pH 6.2 to about pH 7.8, about pH 6.4 to about pH 7.6, about pH 6, 6 to about pH 7.4 or from about pH 6.8 to about pH 7.2. Cells can be cultured in an atmosphere containing from about 10% to about 80%, from about 15% to about 70%, or about 20% to about 60% dissolved oxygen.

The culture may vary depending on the cell type. For cultivation, a known method can be used. The culture may be performed on a plate, flask or the like. Culturing can be done by attaching cells to a substrate or by floating cells in a culture medium. The culture may be a subculture, batch culture, fed culture, perfusion culture, or a combination thereof. During cultivation, the cell culture medium can be periodically replaced with fresh medium. Cells can be cultured for about 1 day or more, about 2 days or more, about 3 days or more, about 4 days or more, about 5 days or more, about 6 days or more, about 1 week or more, about 10 days or more, about 2 weeks or more, about 3 weeks or more, about 1 month or more, from about 1 day to about 1 month, from about 1 day to about 3 weeks, from about 1 day to about 2 weeks, from about 2 days to about 2 weeks, about 3 days to about 2 weeks, about 4 days to about 2 weeks, about 5 days to about 2 weeks, about 6 days to about 2 weeks, or about 1 week to about 2 weeks weeks

It may include obtaining a protein complex based on the first polypeptide, the second polypeptide, or the first polypeptide and the second polypeptide from cells or cell culture medium.

The cell culture medium may be a cell-free culture medium.

When co-transformed with a cell expression vector, a protein complex based on the first polypeptide and the second polypeptide can be obtained from the cells or cell culture medium. If at least two cell types are transformed with an expression vector encoding a first polypeptide and an expression vector encoding a second polypeptide, one vector for each cell type, the first polypeptide and the second polypeptide can be obtained from the cells or cell culture media.

Preparation of the protein complex may include incubating the resulting first polypeptide and the resulting second polypeptide to form a protein complex. Incubation can be performed under reducing condition. The reducing condition can be provided in the presence of 2-mercaptoethanol (2-ME), dithiothreitol (DTT), or a combination thereof.

Preparation of the protein complex may involve purification of the protein complex. Purification can be accomplished by filtration, centrifugation, chromatography, dialysis, immunoprecipitation, or a combination thereof.

In another aspect, a pharmaceutical composition for preventing or treating cancer is provided, comprising a protein complex according to one aspect.

Specific details regarding the protein complex are described above.

The cancer may be a solid cancer or a non-solid cancer. Solid cancer refers to malignant tumors that arise in organs such as the liver, lung, breast, skin, etc. Non-solid cancers are cancers that originate in the blood, also called hematologic malignancies. The cancer may be a carcinoma, sarcoma, cancer derived from hematopoietic cells, germ cell tumor or blastoma. The cancer may be selected from the group consisting of breast cancer, skin cancer, head and neck cancer, pancreatic cancer, lung cancer, colorectal cancer, stomach cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer , kidney cancer, clear cell sarcoma, melanoma, cerebrospinal tumor, brain cancer, thymoma, mesothelioma, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer and sarcoma.

The term "prophylaxis" refers to any action that suppresses a disease or delays the onset of a disease by administering a pharmaceutical composition. The term "treatment" refers to any action that improves or favorably modifies the symptoms of a disease by administering a pharmaceutical composition.

The pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "carrier" is used to cover auxiliaries, diluents or excipients. For example, the carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, physiological saline solution, buffers such as PBS, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. The composition may include a filler, an antiaggregant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, or a combination thereof.

The pharmaceutical composition can be prepared in any composition according to the method of the prior art. The composition may be formulated, for example, as a formulation for oral administration (eg, powder, tablet, capsule, syrup, pill or granule) or a formulation for parenteral administration (eg, by injection). In addition, the composition may be prepared as a formulation for systemic administration or as a formulation for topical administration.

The pharmaceutical composition may further include other anticancer agents. The anticancer agent may be cetuximab, panitumumab, erlotinib, gefitinib, trastuzumab, T-DM1, Perjeta, lapatinib, paclitaxel, taxol, tamoxifen, cisplatin, or a combination thereof. The pharmaceutical composition may be a single composition or separate compositions. For example, a composition based on an antibody or an antigen-binding fragment thereof may be a composition for parenteral administration, and an anticancer agent may be a composition for oral administration.

The pharmaceutical composition may include the protein complex in an effective amount. The term "effective amount" means an amount sufficient to produce a prophylactic or treatment effect when administered to a subject in need of prophylaxis or treatment of a disease. The effective amount can be appropriately selected by those skilled in the art depending on the cell or subject. The effective amount may be determined based on the severity of the disease, age, body weight, medical condition, sex of the patient, sensitivity of the patient to the drug, time of administration, route of administration and rate of elimination, duration of treatment, drug used in combination or concurrently with the composition used , and other factors well known in the medical field. An effective amount may be from about 0.5 μg to about 2 g, from about 1 μg to about 1 g, from about 10 μg to about 500 mg, from about 100 μg to about 100 mg, or from about 1 mg to about 50 mg for a pharmaceutical composition.

The dosage of the pharmaceutical composition for adults may range, for example, from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, or from about 0.1 mg/kg to about 1 mg/kg. Administration can be once a day, several times a day, once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks to once a year.

In another aspect, a method of preventing or treating cancer is provided, comprising administering a protein complex according to one aspect to a cell or subject.

Specific details regarding the protein complex, cells, cancer, prevention or treatment are described above.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat or cat. The subject may be a subject who has cancer or is at risk of cancer.

The method may further comprise administering the second active ingredient to the subject. The second active ingredient may be an active ingredient for preventing or treating cancer. The active ingredient can be administered simultaneously, separately or sequentially with the protein complex.

The protein complex may, for example, be administered directly to a subject by any route, such as oral, intravenous, intramuscular, transdermal, transmucosal, intranasal, intratracheal, or subcutaneous administration. The protein complex can be administered systemically or locally, alone or in combination with other pharmaceutically active compounds.

The effective amount of the protein complex may vary depending on the patient's condition and body weight, severity of the disease, formulation of the drug, route and duration of administration, etc., and can be appropriately selected by those skilled in the art. The dosage may range, for example, from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, or from about 0.1 mg/kg to about 1 mg/kg for adults. The protein complex can be administered once a day, several times a day, once a week, once every 2 weeks, once every 3 weeks, or from once every 4 weeks to once a year.

USEFUL EFFECTS OF THE INVENTION

According to a protein complex characterized by a high rate of heterodimer formation, a method for its preparation, a pharmaceutical composition for preventing or treating cancer including the protein complex, and a method for preventing or treating cancer through its use, bispecific antibodies or antigen-binding fragments with increased stability, receptors and receptor-binding agonists, antagonists, ligands, cytokines or decoy receptor biconjugates can be easily prepared and used in various fields such as disease prevention or treatment and disease diagnosis.

BRIEF DESCRIPTION OF THE DRAWINGS

In fig. 1 is an image showing the results of SDS-PAGE analysis of Fc variants (M: size marker, WT: PTCWT (negative control group), 019: PTC019 (positive control group), 039: PTC039, 040: PTC040, 074: PTC074, 111: RTS111).

In fig. 2 is a graph showing the results of analysis of Fc variants by capillary electrophoresis-SDS (CE-SDS).

In fig. 3A-3F are graphs showing the results of size exclusion high performance liquid chromatography (SE-HPLC) analysis of PTCWT, PTC019, PTC039, PTC040, PTC074 and PTC111, respectively.

In fig. 4 is an image showing the results of SDS-PAGE analysis of Fc variants with reverse sequence substitution (M: size marker, WT: PTCWT (negative control group), 019: PTC019 (positive control group), 088: PTC088, 089: PTC089, 090: RTS090).

In fig. 5 is an image showing the results of SDS-PAGE analysis of Fc variants in which part of the sequence is replaced (M: size marker, WT: PTCWT (negative control group), 019: PTC019 (positive control group), 074: PTC074, 097: RTS097, 098: RTS098, 099: RTS099, 111: RTS111, 113: RTS113).

In fig. 6 is an image showing the results of SDS-PAGE analysis of Fab-Fc × scFv-Fc biconjugates (M: size marker, WT: Fab-Fc × scFv-Fc biconjugate with the PTCWT sequence inserted into the CH3 domain (negative control group), 019: Fab-Fc × scFv-Fc biconjugate with the PTC019 sequence inserted into the CH3 domain (positive control group), 074: Fab-Fc × scFv-Fc biconjugate with the PTC074 sequence inserted into the CH3 domain).

In fig. 7 is an image showing the results of SDS-PAGE analysis of Fab-Fc × scFv-scFv-Fc biconjugates (M: size marker, WT: Fab-Fc × scFv-scFv-Fc biconjugate with the PTCWT sequence inserted into the CH3 domain (group negative control), 019: Fab-Fc × scFv-scFv-Fc biconjugate with the PTC019 sequence inserted into the CH3 domain (positive control group), 074: Fab-Fc × scFv-scFv-Fc biconjugate with the PTC074 sequence inserted into the CH3 -domain).

In fig. 8 is an image showing the results of SDS-PAGE analysis of Fab-Fc × cytokine-Fc biconjugates (M: size marker, WT: Fab-Fc × cytokine-Fc biconjugate with the PTCWT sequence inserted into the CH3 domain (negative control group), 019: Fab-Fc × cytokine-Fc biconjugate with the PTC019 sequence inserted into the CH3 domain (positive control group), 074: Fab-Fc × cytokine-Fc biconjugate with the PTC074 sequence inserted into the CH3 domain).

In fig. 9 shows a graph assessing the ability of bispecific antibodies to PD-L1 × scFv to CD3 to induce cancer cell death in vitro (avelumab: positive control group, BsAb to PD-L1 × scFv to CD3: bispecific antibody to PD-L1 × scFv to CD3, having sequence PTC074 in the CH3 domain).

In fig. 10 shows a graph assessing the ability of bispecific antibodies to PD-L1 × scFv to CD3 to suppress the growth of cancer cells in vivo (avelumab: positive control group, BsAb to PD-L1 × scFv to CD3: bispecific antibody to PD-L1 × scFv to CD3, having sequence PTC074 in the CH3 domain).

PRINCIPLE OF THE INVENTION

The following are preferred examples for a better understanding of the present invention. However, the following examples are provided solely to make the present invention easier to understand, and the scope of the present invention is not limited to the following examples.

Examples

Example 1. Preparation of heterodimeric Fc variants

To evaluate the heterodimer-forming ability of Fc, which changes as a result of amino acid substitution in the CH3 domain of the antibody, an expression system was constructed for each constructed Fc variant.

To facilitate the distinction and analysis of the two Fc polypeptides constituting the heterodimer, the first polypeptide was designed to express the intact form of the IgG chain (Fc1), in which the heavy chain and light chain are linked, and the second polypeptide was designed to express only the heavy chain of the Fc region (Fc2). In Fc1, amino acids in the CH3 domain were replaced based on the antibody avelumab (Bavencio®, Pfizer) comprising the Fc region of wild-type IgG1, and in Fc2, amino acids in the CH3 domain were replaced based on the Fc region of the wild-type IgG1 antibody. The Fc1 light chain has the same amino acid sequence as the light chain of the antibody avelumab (SEQ ID NO: 2).

For comparison, wild-type IgG1 (corresponding to “PTCWT” in Table 1) was used as a negative control group, and a bispecific antibody generated by the knob-in-hollow (KiH) approach from Genentech (corresponding to “PTC019” in Table 1) was used as a positive control group.

Specifically, expression vectors were prepared by introducing the nucleotide open reading frame (ORF) encoding each polypeptide into pCHO1.O vectors. The ExpiCHO-S™ cell line (Thermo Fisher) was cultured in ExpiCHO™ expression medium. Fc1 expression vectors and Fc2 expression vectors were mixed in a 1:1 ratio and transfected into the ExpiCHO-S™ cell line using the ExpiFectamine™ CHO Transfection Kit (Thermo Fisher). After culturing the transfected cells in expression medium, the culture medium was separated and removed. The recovered culture medium was purified using a Protein A HP SpinTrap™ column (GE Healthcare). For purified protein, the buffer was replaced with PBS (pH 7.4) and the protein concentration was measured.

The expressed amino acid sequences were as described below.

[PTCWT]

Heavy chain from Fc1:

(bold: L351, bold and underlined: T366).

Light chain from Fc1:

Fc chain from Fc2:

(bold: L351, bold and underlined: T366, underlined: L368, italic: Y407).

[RTS019]

Heavy chain from Fc1:

(bold: L351, bold and underlined: T366W).

Light chain from Fc1 (SEQ ID NO: 2)

Fc chain from Fc2:

(bold: L351, bold and underlined: T366S, underlined: L368A, italic: Y407V).

[RTS039]

Heavy chain from Fc1:

(bold: L351F, bold and underlined: T366W).

Light chain from Fc1 (SEQ ID NO: 2)

Fc chain from Fc2:

(bold: L351G, bold and underlined: T366S, underlined: L368A, italic: Y407V).

[RTS040]

Heavy chain from Fc1:

(bold: L351W, bold and underlined: T366W).

Light chain from Fc1 (SEQ ID NO: 2)

Fc chain from Fc2 (SEQ ID NO: 7)

[PTC074]

Heavy chain from Fc1 (SEQ ID NO: 4)

Light chain from Fc1 (SEQ ID NO: 2)

Fc chain from Fc2 (SEQ ID NO: 7)

[PTC111]

Heavy chain from Fc1 (SEQ ID NO: 4)

Light chain from Fc1 (SEQ ID NO: 2)

Fc chain from Fc2:

(bold: L351F, bold and underlined: T366S, underlined: L368A, italic: Y407V).

Example 2. Comparison of heterodimer-forming abilities of Fc variants

Using the transient expression system constructed in Example 1 above, the ability to form Fc heterodimers that are altered by amino acid substitution in the CH3 domain was compared, thereby selecting for highly efficient heterodimeric Fc variants.

As an evaluation method, after performing SDS-PAGE under non-reducing conditions, the intensity of the PAGE bands corresponding to the heterodimers was measured and compared.

Specifically, the protein purified in Example 1 was reduced with 2-mercaptoethanol or the sample was prepared without treatment with 2-mercaptoethanol. The reduced or unreduced sample was electrophoresed by SDS-PAGE and the intensity of the electrophoretic band was measured using the ChemiDoc™ Imaging System (Bio-Rad) and Image Lab™ Software (Bio-Rad).

The ability to form Fc heterodimers according to amino acid substitution in the CH3 domain was calculated taking into account the measured band intensities, and the results are presented in Table 1 below.

As a result, as shown in Table 1, the heterodimer formation rates of the negative control group (PTCWT) and the positive control group (PTC019) were 41.5% and 64.6%, respectively, while the heterodimer formation rates of PTC032, PTC033 were confirmed to be , RTS034, RTS037, RTS039, RTS040, RTS061, RTS074, RTS082, RTS091, RTS111 and RTS113 were 78.7%, 76.4%, 76.1%, 85.8%, 93.8%, 94.6% , 70.9%, 94.9%, 79.3%, 75.4%, 92.7% and 82.8%, respectively. In particular, it was confirmed that for PTC039, PTC040, PTC074 and PTC111 the ratio of Fc heterodimers exceeded 90%.

That is, the Fc variant, in one aspect, may exhibit superior ability to form Fc heterodimers.

Example 3. Evaluation of heterodimer-forming abilities of highly effective Fc variants

3-1. Analysis by SDS-PAGE

To evaluate the heterodimer-forming ability of the high-potency Fc variants selected in Example 2, SDS-PAGE analysis was performed in the same manner as in Example 2.

In particular, the intensities of the electrophoretic bands obtained from SDS-PAGE analysis were measured. Thereafter, the formation rates of Fc heterodimers according to the amino acid substitution in the CH3 domain were confirmed by the intensity of the measured bands, and the results are presented in Table 2 below (N/A: not applicable).

In fig. 1 is an image showing the results of analysis of Fc variants by SDS-PAGE.

As a result, as shown in Figure 1, it was confirmed that the intensity of the band corresponding to the Fc1-1 homodimer (146.8 kDa) of the selected PTC039, PTC040, PTC074 and PTC111 was noticeably reduced compared with the negative control group (PTCWT). and the intensity of the band corresponding to the Fc2 monomer (25.9 kDa) was markedly reduced compared to the positive control group (PTS019). On the other hand, it was confirmed that the Fc1-2 heterodimer was formed with high efficiency at 99.3 kDa.

In addition, as shown in Table 1, in the cases of the negative control group (PTCWT) and positive control group (PTC019), it was confirmed that not only Fc1-2 heterodimer but also Fc1-1 homodimer was formed with a rate of 17.1% in the group negative control (PTCWT), and it was confirmed that Fc-2-2 homodimer was formed at a rate of 39.9% in the negative control group (PTCWT) and 13.3% in the positive control group (PTC019). On the other hand, in the cases of the selected Fc variants PTC039, PTC040, PTC074 and PTC111, it was confirmed that the formation rate of Fc1-1 homodimers and Fc-2-2 homodimers was low, and the formation rate of Fc1-2 heterodimers was noticeably higher.

That is, it can be seen that the Fc variant, according to one aspect, is capable of selectively forming Fc1-2 heterodimers.

3-2. Analysis by CE-SDS

In order to test the heterodimer-forming abilities of the high-potency Fc variants selected in Example 2, capillary electrophoresis-SDS (CE-SDS) analysis was performed.

Specifically, capillary electrophoresis was performed using the PA 800 plus™ Pharmaceutical Analytical System (SCIEX) to test the Fc heterodimer formation rates of the Fc variants, and the results are shown in Table 3 below.

In fig. 2 is a graph showing the results of analysis of Fc variants by capillary electrophoresis-SDS (CE-SDS).

As a result, as shown in FIG. 2, with a retention time of about 23 minutes, the formation rate of Fc-2-2 homodimers in the negative control group (PTCWT) was 34.44%, and the formation rate of Fc-2-2 homodimers in the positive control group (PTC019) was 13.71% . On the other hand, in the cases of PCT039, PTC040 and PTC074, no Fc-2-2 homodimer was formed, and in the case of PCT111, the formation rate of Fc-2-2 homodimers was 0.57%, and this rate was noticeably lower compared to the negative control group (PTCWT) and a positive control group (PCT019).

In addition, as shown in Table 2, the heterodimer formation rates of PTC039, PTC040, PTC074, and PTC111 were 81.0%, 80.2%, 83.8%, and 84.8%, respectively, and they were significantly higher than that of negative control group (PCTWT) and positive control group (RTC019).

That is, it can be seen that the Fc variant according to one aspect has excellent heterodimer-forming ability.

3-3. Analysis by SE-HPLC

In order to test the heterodimer-forming abilities of the high performance Fc variants selected in Example 2, size exclusion high performance liquid chromatography (SE-HPLC) analysis was performed and the results are shown in Table 4 below.

In fig. 3A-3F are graphs showing the results of size exclusion high performance liquid chromatography (SE-HPLC) analysis of PTCWT, PTC019, PTC039, PTC040, PTC074 and PTC111, respectively.

As a result, as shown in FIG. 3A-3F, in SE-HPLC analysis of Protein A purified protein, major heterodimer peaks were observed at retention times ranging from 16.989 minutes to 17.197 minutes in Fc variants including a negative control group (PTCWT) and a positive control group (PTC019) . Specifically, in the case of the negative control group (PTCWT), the Fc-1-1 homodimer peak was observed at a retention time of 16.079 minutes, and the Fc2-2 homodimer peak was observed at 18.377 minutes (Fig. 3A). Moreover, in the case of the positive control group (PTC019), the Fc-2-2 homodimer peak was observed at a retention time of 18.343 minutes, and the Fc-2 monomer peak was observed at 19.14 minutes (Figure 3B). That is, in the case of the negative control group (PTCWT) and the positive control group (PCT019), the heterodimer formation rate was confirmed to be low, since several peaks including the Fc1-2 heterodimer were shown, on the other hand, in the case of the Fc variants PTC039, PTC040, PTC074 and PTC111 the main peak was observed as a separate peak. That is, in the case of the selected Fc variants, the heterodimer formation rate was confirmed to be superior compared to the negative control group (PTCWT) and the positive control group (PTC019).

In addition, as shown in Table 4, it was confirmed that the selected Fc variants PTC039, PTC040, PTC074 and PTC111 had better heterodimer formation abilities compared with the two control groups PTCWT and PTC019.

Thus, the Fc variant in one aspect can form a heterodimer with high efficiency, and therefore bispecific antibodies can be easily produced.

Example 4. Evaluation of the thermodynamic stability of heterodimers of highly efficient Fc variants

To indirectly confirm the structural stabilities of the heterodimers of high-potency Fc variants identified in Example 3, thermodynamic stability was assessed using differential scanning calorimetry (DSC).

In particular, the thermal stabilities of the selected Fc variants were measured using Nano DSC™ (TA instruments) and melting temperatures (Tm, °C) were calculated. The calculated melting points of the Fc variants are shown in Table 5 below.

As a result, as shown in Table 5, it was confirmed that the selected Fc variants PTC039, PTC040, PTC074 and PTC111 had slightly unstable thermal stability compared to the wild type Fc heterodimer (PTCWT), but had relatively high thermodynamic stability compared to the heterodimer Fc KiH (RTC019) from Genentech.

That is, the Fc variant according to one aspect is characterized by excellent heterodimer-forming ability as well as excellent thermodynamic stability, so that stable bispecific antibodies can be formed.

Example 5. Comparison of heterodimer-forming abilities of variants substituted with a reverse sequence in the CH3 domain among highly efficient Fc variants

To evaluate the heterodimer-forming abilities of variants substituted with a reverse sequence in the CH3 domain, among the high-potency Fc variants selected in Example 3 above, SDS-PAGE analysis was performed in the same manner as in Example 2.

The Fc heterodimer formation abilities according to the amino acid substitution in the CH3 domain were calculated taking into account the band intensities measured in the SDS-PAGE analysis, and the results are presented in Table 6 below.

In fig. 4 is an image showing the results of SDS-PAGE analysis of Fc variants with reverse sequence substitution (M: size marker, WT: PTCWT (negative control group), 019: PTC019 (positive control group), 088: PTC088, 089: PTC089, 090: RTS090).

As a result, as shown in FIG. 4, it was confirmed that the Fc variants with reverse sequence substitution did not form a band around 150 kDa compared to the negative control group (PTCWT), and the bands were weakly formed at 49.2 kDa. That is, it was confirmed that Fc variants with reverse sequence substitution did not form the Fc-1-l homodimer, and the formation of Fc-2-2 homodimers was reduced. In addition, it was confirmed that, compared with the positive control group (PCT019), a band of about 25 kDa was weakly formed. That is, it can be seen that Fc variants with a reverse sequence substitution are characterized by reduced formation of Fc-2 monomers and exhibit rates of heterodimer formation similar to PTC039, PTC040 and PTC074 (see Fig. 1).

In addition, as shown in Table 6, the heterodimer formation rates of the negative control group (PTCWT) and positive control group (PTC019) were 40.9% and 64.7%, respectively, while PTC088, PTC089 and PTC090 were confirmed to be which are Fc variants with reverse sequence substitution PTC039, PTC040 and PTC074 showed heterodimer formation rates of 72.1%, 83.3% and 92.1%, respectively.

Thus, it can be seen that variants with a substitution of the reverse sequence of the Fc variant according to one aspect form a heterodimer at a similar ratio before and after the substitution. In addition, since the reverse sequence substitution variants of the Fc variant have an excellent heterodimer formation rate, Fc variants can be formed with high efficiency regardless of whether the sequence inserted in Fc1 or Fc2 is substituted or not.

Example 6. Comparison of heterodimer-forming abilities of Fc variants with a sequence changed at position 351 of the CH3 domain

To evaluate the heterodimer-forming ability of variants in which position 351 in the CH3 domain is replaced by an amino acid having similar properties to PTC074 and PTC111, which are the highly efficient Fc variants selected in Example 3, SDS-PAGE analysis was performed in the same manner. , as in example 2.

The heterodimer-forming abilities of Fc according to amino acid substitution in the CH3 domain were calculated taking into account the band intensities measured by SDS-PAGE analysis, and the results are presented in Table 7 below.

In fig. 5 is an image showing the results of SDS-PAGE analysis of Fc variants in which part of the sequence is replaced (M: size marker, WT: PTCWT (negative control group), 019: PTC019 (positive control group), 074: PTC074, 097: RTS097, 098: RTS098, 099: RTS099, 111: RTS111, 113: RTS113).

As a result, as shown in FIG. 5, in the cases of PTC074 and PTC111, it was confirmed that no band around 150 kDa was produced compared to the negative control group (PTCWT), and compared to the positive control group (PTC019), the intensity of the bands at 49.2 kDa and 24.6 kDa was noticeable reduced. In addition, it was confirmed that the intensity of the bands at 24.6 kDa was markedly reduced compared to PTC097, PTC098 and PTC099, in which the sequence was replaced at position 351 in Fc-2. On the other hand, it was confirmed that the band intensity increased at 98 kDa, which is the calculated molecular weight of the Fc-1-2 heterodimer.

In addition, as shown in Table 7, the heterodimer formation rates of PTC074, PTC097, PTC099, and PTC111 were 93.5%, 82.7%, 86.5%, and 92.7%, respectively, and confirmed that they were significantly higher than that of the negative control group (PCTWT) and the positive control group (RTC019). In particular, in the cases of PTC074 and PTC111, in which position 351 in the CH3 domain was replaced by glycine (G) or phenylalanine (F), the formation rate of Fc heterodimers was confirmed to be greater than 90%.

That is, it can be seen that the Fc variant according to one aspect has an excellent ability to form Fc heterodimers by replacing position 351 in the CH3 domain with a specific amino acid.

Example 7. Evaluation of the heterodimer-forming ability of bispecific antibodies, including highly effective Fc variants

7-1. Bispecific antibody Fab-Fc × scFv-Fc

To evaluate the heterodimer formation rate of a heterobispecific antibody comprising the highly potent Fc variants selected in Example 3, the first polypeptide was designed to express the intact form of the IgG chain (Fc1), in which the heavy chain and light chain are linked, and the second polypeptide was designed to express the scFV form -Fc (Fc2). Fc1 was then expressed using the antibody avelumab (Bavencio, Pfizer) comprising the IgG1 Fc region, and Fc2 was expressed as a scFv comprising the IgG1 Fc region based on the anti-CD3 scFv sequence of blinatumomab.

For comparison, the heterobispecific antibody Fab-Fc × scFv-Fc, in which the anti-CD3 scFv of blinatumomab is linked to Fc2 from the Fc variant having the wild-type CH3 domain sequence (corresponding to “PTCWT” in Table 1), and As a positive control group, we used the heterobispecific antibody Fab-Fc × scFv-Fc, in which the anti-CD3 scFv of blinatumomab is linked to Fc2 from the Fc variant (corresponding to “RTC019” in Table 1) having the sequence in the CH3 domain obtained by the " ridges-in-hollows" (KiH) from Genentech.

Expression of each test substance was carried out using the transient expression system constructed in Example 1, and after performing SDS-PAGE under non-reducing conditions, the intensities of the PAGE bands corresponding to the heterodimers were measured to compare rates of heterodimer formation.

The ability to form an Fc heterodimer according to amino acid substitution in the CH3 domain was calculated from the measured band intensities and the results are presented in Table 8 below.

In fig. 6 is an image showing the results of SDS-PAGE analysis of Fab-Fc × scFv-Fc biconjugates (M: size marker, WT: Fab-Fc × scFv-Fc biconjugate with the PTCWT sequence inserted into the CH3 domain (negative control group), 019: Fab-Fc × scFv-Fc biconjugate with the PTC019 sequence inserted into the CH3 domain (positive control group), 074: Fab-Fc × scFv-Fc biconjugate with the PTC074 sequence inserted into the CH3 domain).

As a result, as shown in FIG. 6 confirmed that the heterobispecific antibody Fab-Fc × scFv-Fc into which the PTC074 sequence was inserted did not produce a band at 144 kDa, in contrast to the heterobispecific antibody into which the sequence from the negative control group (PTCWT) was inserted. In addition, it was confirmed that the band intensity was reduced at 104.8 kDa compared to the heterobispecific antibody into which the PTC019 sequence was inserted. On the other hand, at 124.4 kDa, which is the calculated molecular weight of the complete Fab-Fc × scFv-Fc heterobispecific antibody, it was confirmed that the band intensity was increased compared with the negative control group (PCTWT) and positive control group (PTC019) .

In addition, as shown in Table 8, the Fc1-2 heterodimer formation rate of the heterobispecific antibody Fab-Fc × scFv-Fc, which contains the PTC074 CH3 domain sequence, was 88.8%, which confirmed that the heterodimer formation rate was significantly higher than that of the negative control group (PTCWT) and positive control group (PTC019).

That is, it can be seen that the Fc variant according to one aspect can be used as a structure for a heterobispecific antibody that produces antibodies of different structures and has excellent heterodimer formation ability.

7-2. Bispecific antibody Fab-Fc × scFv-scFv-Fc

Heterodimer formation rates were assessed in the same manner as in Example 7-1, except that Fab-Fc × scFv-scFv-Fc bispecific antibodies designed to express a second polypeptide in the form scFv-scFv-Fc (Fc2) were used. a Fc2 was expressed as a dual scFv (scFv × scFv) comprising the IgG1 Fc region, based on the anti-CD3 scFv sequence of blinatumomab and the VH-VL sequence of bevacizumab (Avastin, Roche).

For comparison, the heterobispecific antibody Fab-Fc × scFv-scFv-Fc, in which the double scFv is linked to Fc2 from the Fc variant having the wild-type CH3 domain sequence (corresponding to “PTCWT” in Table 1), and As a positive control group, the heterobispecific antibody Fab-Fc × scFv-scFv-Fc was used, in which the double scFv is associated with Fc2 from the Fc variant (corresponding to “PTS019” in Table 1) having the sequence in the CH3 domain obtained by the " ridges-in-hollows" (KiH) from Genentech.

In fig. 7 is an image showing the results of SDS-PAGE analysis of Fab-Fc × scFv-scFv-Fc biconjugates (M: size marker, WT: Fab-Fc × scFv-scFv-Fc biconjugate with the PTCWT sequence inserted into the CH3 domain (group negative control), 019: Fab-Fc × scFv-scFv-Fc biconjugate with the PTC019 sequence inserted into the CH3 domain (positive control group), 074: Fab-Fc × scFv-scFv-Fc biconjugate with the PTC074 sequence inserted into the CH3 -domain).

As a result, as shown in FIG. 7, it was confirmed that the Fab-Fc × scFv × scFv-Fc biconjugate into which the PTC074 sequence was inserted had a markedly reduced band intensity corresponding to the size of 196 kDa compared to the conjugate into which the positive control group (PTCWT) sequences were inserted ) and positive control group (RTS019). In addition, since the band intensities were reduced except for the heterodimer band shown in the biconjugate into which the positive control group sequence (PTC019) was inserted, it was confirmed that the target 167.8 kDa Fc 1-2 heterodimer was obtained with high efficiency.

In addition, as shown in Table 9, the Fc1-2 heterodimer formation rate of the Fab-Fc×scFv-scFv-Fc heterobispecific antibody containing the CH3 domain sequence of PTC074 was 70.2%, and it was confirmed that the heterodimer formation rate was significantly higher than that of the negative control group (PTCWT) and positive control group (PTC019).

That is, it can be seen that the Fc variant according to one aspect not only can be easily used as a structure of a heterobispecific antibody having a large molecular weight structure since multiple structures are connected, but it also has excellent heterodimer formation when used as a structure.

7-3. Bispecific antibody Fab-Fc × cytokine-Fc

Heterodimer formation rates were assessed in the same manner as in Example 7-1, except that Fab-Fc × cytokine-Fc bispecific antibodies engineered to express a second polypeptide in the form of cytokine-Fc (Fc2) were used and Fc2 was expressed in the form of a cytokine comprising the Fc region of IgG1, which is based on the sequence of interleukin-2 (IL-2, aldesleukin).

For comparison, the heterobispecific antibody Fab-Fc × IL-2v-Fc, in which the IL-2 variant (IL-2v) is linked to Fc2 from the Fc variant having the wild-type CH3 domain sequence (corresponding to “PTCWT”), was used as a negative control group " in Table 1), and the heterobispecific antibody Fab-Fc × IL-2v-Fc, in which the IL-2 variant (IL-2v) is linked to Fc2 from the Fc variant (corresponding to "RTC019" in Table 1), was used as a positive control group 1) having a sequence in the CH3 domain obtained through the knobs-to-holes (KiH) approach from Genentech.

In fig. 8 is an image showing the results of SDS-PAGE analysis of Fab-Fc × cytokine-Fc biconjugates (M: size marker, WT: Fab-Fc × cytokine-Fc biconjugate with the PTCWT sequence inserted into the CH3 domain (negative control group), 019: Fab-Fc × cytokine-Fc biconjugate with the PTC019 sequence inserted into the CH3 domain (positive control group), 074: Fab-Fc × cytokine-Fc biconjugate with the PTC074 sequence inserted into the CH3 domain).

As a result, as shown in FIG. 8, it was confirmed that the Fab-Fc × cytokine-Fc biconjugate, into which the sequences PTC074 and PTC111 were inserted, was characterized by a reduced intensity of the band corresponding to the size of 94.4 kDa (Fc-2-2 homodimer) and 77.4 kDa (monomer Fc1), and increased intensity corresponding to the size of 136.5 kDa (heterodimer Fc-1-2), compared with the negative control group (PTCWT) or the positive control group (PTC019). Thus, in the Fab-Fc × cytokine-Fc biconjugate into which the sequences PTC074 and PTC111 were inserted, the formation of the band corresponding to the Fc-2-2 homodimer and Fc-1 monomers was reduced, and thus it can be seen that Fc-1-2 heterodimers were formed with high efficiency.

In addition, as shown in Table 10, according to the structure in which the sequences of the negative control group (PTCWT), positive control group (PTC019), PTC074 and PTC111 are present in the Fab-Fc × cytokine-Fc biconjugate, the ratio of Fc-1-2 heterodimers were 40.6%, 68.8%, 90.4% and 89.9%, respectively, and it was confirmed that biconjugates exhibiting PTC074 and PTC111 had a significantly higher heterodimer formation rate compared to the negative control group (PTCWT) and a positive control group (RTS019).

That is, it can be seen that the Fc variant according to one aspect can be used as a structure that forms an antibody, and when a cytokine protein is used, the heterodimer formation ability is excellent.

7-4. Bispecific antibody Fab-Fc × Fab-Fc

To evaluate the heterodimer formation rates of heterobispecific antibodies comprising the highly potent Fc variants selected in Example 3, both the first polypeptide and the second polypeptide were designed to express bispecific antibodies in an intact form in which the heavy and light chains are linked. Specifically, Fc1 was expressed based on the antibody avelumab (Bavencio, Pfizer) comprising the Fc region of wild-type IgG1, and Fc2 was expressed based on the antibody bevacizumab (Avastin, Roche) comprising the Fc region of IgG1.

For comparison, a Fab-Fc × Fab-Fc heterobispecific antibody including an Fc variant having a wild-type CH3 domain sequence (corresponding to “PTCWT” in Table 1) was used as a negative control group, and a Fab heterobispecific antibody was used as a positive control group. -Fc × Fab-Fc, comprising an Fc variant (corresponding to “PTC019” in Table 1) having a sequence in the CH3 domain obtained through the knobs-to-holes (KiH) approach from Genentech.

The Intact MASS analysis method (ESI-LC-MS) was used as the evaluation method. Specifically, liquid chromatography-mass spectrometry (LC-MS) was performed using a Thermo Scientific Dionex™ UHPLC Ultimate 3000 and TripleTOF 5600+ (AB sciex), and Analyst® and PeakView® Software were used as analysis programs for heterodimer calculations -forming capacity, and the results are presented in Table 11 below.

As shown in Table 11, it was confirmed that the Fc-1-2 heterodimer formation rates of Fab-Fc×Fab-Fc heterobispecific antibodies into which the negative control group (PTCWT) and positive control group (PTC019) sequences were inserted were 25 .4% and 32.4%, respectively, on the other hand, the formation rates of the Fc-1-2 heterodimer in heterobispecific antibodies having the sequences PTC039, PTC040 and PTC074 of the CH3 domain were 58.1%, 40.5% and 46. 8% respectively.

That is, it can be seen that the Fc variant according to one aspect not only can be easily used as a bispecific antibody, but it also has excellent heterodimer formation when used as the above-mentioned structure.

Example 8. Evaluation of anticancer activity of bispecific antibodies Fab-Fc × scFv-Fc, including highly effective Fc variants

8-1. Confirmation of cancer cell death and suppression of cancer cell growth

The anti-PD-L1 x anti-CD3 scFv bispecific antibody produced in Example 7-1 was evaluated for its ability to kill cancer cells and inhibit cancer cell growth.

Specifically, 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin mixture were added to RPMI1640 medium (#11875-093, Gibco), and breast cancer cells (MDA-MB- 231) were cultured at 37°C in an atmosphere of 5% CO ₂ . After cell division in a 96-well plate at a concentration of 10,000 cells/well, the cells were cultured overnight at 37°C in an atmosphere of 5% CO ₂ , and human peripheral blood mononuclear cells (PBMCs) (effector cells) were co-cultured after division in the ratio 20:1 (effector cells:target cells (MDA-MB-231)). Cells in the wells were then treated with anti-PD-L1 × anti-CD3 scFv bispecific antibodies and then cultured for 48 hours. After recovery of the supernatant, cytotoxicity was then assayed according to Equation 1 below using the LDH Cytotoxicity Assay Kit (#C20301, Invitrogen). Avelumab (anti-PD-L1 antibody, Bavencio®) was used as a positive control group.

In fig. 9 shows a graph assessing the ability of bispecific antibodies to PD-L1 × scFv to CD3 to induce cancer cell death in vitro (avelumab: positive control group, BsAb to PD-L1 × scFv to CD3: bispecific antibody to PD-L1 × scFv to CD3, having sequence PTC074 in the CH3 domain).

As a result, as shown in FIG. 9, the Emax (maximum cytotoxic effect) in the positive control group was 26% at the maximum concentration of the substance, while the Emax for the heterobispecific antibody Fab-Fc × scFv-Fc was 76.3%, which confirmed that the heterobispecific antibody has a markedly high cytotoxic effect against cancer cells. Additionally, the positive control group exhibited saturation with no further increase in cell lysis at 2 _log ng/mL or greater, confirming that the effective amount was produced in the range of 0 _log ng/mL to 2 _log ng/mL. On the other hand, the heterobispecific antibody Fab-Fc × scFv-Fc showed saturation with no further increase in cell lysis at drug concentrations of 1 _log ng/ml or greater, confirming that the effective amount was formed in the range of -1 _log ng/ml to 1 _log ng/ml.

In addition, as shown in Table 12, in the case of the positive control group, the cancer cell growth suppression effect of MDA-MB-231 was represented by the EC value₅₀ 11.8 ng/ml, while the heterobispecific antibody Fab-Fc × scFc-Fc was characterized by EC₅₀ 1.3 ng/ml, which confirmed that the heterobispecific antibody was more effective in inhibiting the growth of cancer cells.

That is, the Fab-Fc × scFv-Fc heterobispecific antibody generated by the Fc variant according to one aspect exhibits excellent anticancer effect at a lower concentration compared with known immunotherapy drugs, and the side effects of existing therapeutic agents such as destruction of healthy cells or resistance.

8-2. Confirmation of anti-cancer effectiveness

The in vivo anticancer efficacy of anti-PD-L1 × anti-CD3 scFv bispecific antibody was assessed using a humanized tumor xenograft mouse model. Specifically, a mixture of MDA-MB-231 cells (5 x 10 ⁶ ) and purified human T cells (2.5 x 10 ⁶ ) was mixed with the same amount of Matrigel and injected subcutaneously into the flank of NOD/SCID mice (female, age 6 weeks, Jabio) at a dose of 0.2 ml/mouse. One hour after inoculation, 2.5 mg/kg of the anti-PD-L1 x CD3 scFv bispecific antibody prepared in Example 7-1 was administered intravenously (5 times/week). Avelumab, a positive control, was administered intravenously (3 times/week) at a dose of 20 mg/kg. 7 days after inoculation, tumor volumes were measured (3 times/week) to compare and evaluate the anticancer efficacy of the two substances.

In fig. 10 shows a graph assessing the ability of bispecific antibodies to PD-L1 × scFv to CD3 to suppress the growth of cancer cells in vivo (avelumab: positive control group, BsAb to PD-L1 × scFv to CD3: bispecific antibody to PD-L1 × scFv to CD3, having sequence PTC074 in the CH3 domain).

As a result, as shown in FIG. 10, in the case of the negative control group, the average tumor volume gradually increased over 28 days until it reached 600 mm ³ or more, which confirmed that there was no tumor growth suppression effect. Moreover, in the case of the positive control group, the tumor volume continued to increase, which confirmed that the tumor growth suppression effect was 59% on day 28. On the other hand, in the case of Fab-Fc × scFv-Fc, tumor regression due to tumor growth suppression remained the same same from days 9 to 21, with a tumor growth suppression effect of 80% on day 28, confirming that the bispecific antibody was effective in reducing tumor growth in humanized xenograft tumor mouse models.

That is, the Fab-Fc×scFv-Fc heterobispecific antibody generated by the Fc variant according to one aspect has an excellent tumor growth suppression effect, and thus can be used for the prevention or treatment of various types of cancer, including breast cancer.

The above description of the present invention is presented for illustrative purposes, and those skilled in the art to which the present invention relates will appreciate that the examples and embodiments can be easily modified without changing the technical idea or essential features of the present invention. It should therefore be noted that the above examples are not limiting, but rather illustrative of all aspects.

--->

LIST OF SEQUENCES

<110> MUSTBIO Co., Ltd.

<120> BISPECIFIC ANTIBODY OR ITS ANTIGEN-BINDING FRAGMENT AND METHOD

THEIR RECEIPT

<130>PX064553PCT

<150> KR10-2020-0066030

<151> 2020-06-04

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<---

Claims (44)

1. A protein complex having an increased ability to form an Fc heterodimer compared to its wild type or knob-to-hole (KiH) type, containing a first polypeptide comprising the first CH3 domain of the constant region of a human IgG1 antibody; and a second polypeptide comprising a second CH3 domain of a human IgG1 antibody constant region, wherein the first polypeptide and the second polypeptide form a heterodimer; wherein the first CH3 domain of the human IgG1 antibody constant region contains tryptophan (W) at position 366, and the second CH3 domain of the human IgG1 antibody constant region contains serine (S) at position 366, alanine (A) at position 368 and valine (V ) in position 407; And at least one of the first CH3 domain of a human IgG1 antibody constant region and the second CH3 domain of a human IgG1 antibody constant region contains at least one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), histidine (H ), glycine (G), valine (V), methionine (M) and alanine (A), at one or more positions selected from the group consisting of positions 351 and 394, where the amino acid position corresponds to the EU index according to Kabat. 2. The protein complex according to claim 1, where the first CH3 domain of the constant region of the human IgG1 antibody contains at least one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), histidine (H), glycine (G ), valine (V), methionine (M) and alanine (A), at one or more positions selected from the group consisting of positions 351 and 394. 3. The protein complex according to claim 1, wherein the second CH3 domain of the human IgG1 antibody constant region contains at least one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), histidine (H), glycine (G ), valine (V), methionine (M) and alanine (A), at one or more positions selected from the group consisting of positions 351 and 394. 4. The protein complex according to claim 1, wherein the first CH3 domain of the human IgG1 antibody constant region contains tryptophan (W) at position 366 and phenylalanine (F), histidine (H) or tryptophan (W) at position 394. 5. The protein complex according to claim 1, wherein the first CH3 domain of the human IgG1 antibody constant region contains tryptophan (W) at position 366 and valine (V), tryptophan (W), alanine (A) or phenylalanine (F) at position 351 . 6. The protein complex according to claim 1, where the second CH3 domain of the human IgG1 antibody constant region contains serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407 and alanine (A), glycine (G), valine (V), methionine (M), or phenylalanine (F) at position 351. 7. The protein complex according to claim 1, wherein the first CH3 domain of the human IgG1 antibody constant region contains one selected from the group consisting of: tryptophan (W) at position 366; tryptophan (W) at position 366 and histidine (H) at position 394; tryptophan (W) at position 366 and phenylalanine (F) at position 394; tryptophan (W) at position 366 and tryptophan (W) at position 394; tryptophan (W) at position 366 and phenylalanine (F) at position 351; tryptophan (W) at position 366 and tryptophan (W) at position 351; tryptophan (W) at position 366 and valine (V) at position 351; tryptophan (W) at position 366 and alanine (A) at position 351; And tryptophan (W) at position 366, histidine at position 394 (H), and phenylalanine at position 351 (F). 8. The protein complex according to claim 1, wherein the second CH3 domain of the human IgG1 antibody constant region contains one selected from the group consisting of: serine (S) at position 366, alanine (A) at position 368, and valine (V) at position 407; serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and alanine (A) at position 351; serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and glycine (G) at position 351; serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and valine (V) at position 351; serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and methionine (M) at position 351; And serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and phenylalanine (F) at position 351. 9. The protein complex according to claim 1, where the first CH3 domain of the human IgG1 antibody constant region contains tryptophan (W) at position 366 and phenylalanine (F), histidine (H) or tryptophan (W) at position 394; and the second CH3 domain of the human IgG1 antibody constant region contains a serine (S) at position 366, an alanine (A) at position 368, and a valine (V) at position 407. 10. The protein complex according to claim 1, where the first CH3 domain of the constant region of the human IgG1 antibody contains tryptophan (W) at position 366 and phenylalanine (F) at position 351; and the second CH3 domain of the human IgG1 antibody constant region contains serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and alanine (A) at position 351. 11. The protein complex of claim 10, wherein the first CH3 domain of the human IgG1 antibody constant region further contains a histidine (H) at position 394. 12. The protein complex according to claim 1, where the first CH3 domain of the constant region of the human IgG1 antibody contains tryptophan (W) at position 366; and the second CH3 domain of the human IgG1 antibody constant region contains serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and glycine (G) at position 351. 13. The protein complex of claim 12, wherein the first CH3 domain of the human IgG1 antibody constant region further contains phenylalanine (F) or tryptophan (W) at position 351. 14. The protein complex according to claim 1, where the first CH3 domain of the constant region of the human IgG1 antibody contains tryptophan (W) at position 366; and the second CH3 domain of the human IgG1 antibody constant region contains serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and alanine (A), valine (V), or methionine (M) at position 351. 15. The protein complex according to claim 1, where the first CH3 domain of the constant region of the human IgG1 antibody contains tryptophan (W) at position 366; and the second CH3 domain of the human IgG1 antibody constant region contains serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and phenylalanine (F) at position 351. 16. The protein complex according to claim 1, where the first CH3 domain of the constant region of the human IgG1 antibody contains tryptophan (W) at position 366 and valine (V) at position 351; and the second CH3 domain of the human IgG1 antibody constant region contains serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and alanine (A) at position 351. 17. The protein complex according to claim 1, where the first CH3 domain of the constant region of the human IgG1 antibody contains tryptophan (W) at position 366 and alanine (A) at position 351; and the second CH3 domain of the human IgG1 antibody constant region contains serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and phenylalanine (F) at position 351. 18. The protein complex of claim 1, wherein the first polypeptide and/or the second polypeptide further comprise any one selected from the group consisting of an antigen binding fragment (Fab), a single chain variable fragment (scFv), a membrane receptor extracellular domain, an agonist, an antagonist, a ligand , decoy receptor, cytokine, coagulation factor and affinity tag. 19. A protein complex having an increased ability to form an Fc heterodimer compared to its wild type or knob-to-hole (KiH) type, comprising a first polypeptide comprising a first CH3 domain of a human IgG1 antibody constant region; and a second polypeptide comprising a second CH3 domain of a human IgG1 antibody constant region, wherein the first polypeptide and the second polypeptide form a heterodimer, wherein the first CH3 domain of the constant region of the human IgG1 antibody contains serine (S) at position 366, alanine (A) at position 368 and valine (V) at position 407, the second CH3 domain of the constant region of the human IgG1 antibody contains tryptophan (W) at position 366, and at least one of the first CH3 domain of a human IgG1 antibody constant region and the second CH3 domain of a human IgG1 antibody constant region contains at least one amino acid selected from the group consisting of phenylalanine (F), tryptophan (W), histidine (H ), glycine (G), valine (V), methionine (M) and alanine (A), at one or more positions selected from the group consisting of positions 351 and 394, where the amino acid position corresponds to the EU index according to Kabat. 20. The protein complex of claim 19, wherein the first CH3 domain of the human IgG1 antibody constant region contains serine (S) at position 366, alanine (A) at position 368, valine (V) at position 407, and glycine (G) at position 351; and the second CH3 domain of the human IgG1 antibody constant region contains tryptophan (W) at position 366. 21. The protein complex according to claim 20, wherein the second CH3 domain of the human IgG1 antibody constant region further contains phenylalanine (F) or tryptophan (W) at position 351. 22. A method for producing a protein complex, comprising transforming one or more cells using one or more expression vectors encoding the first polypeptide according to claim 1 or 19, the second polypeptide according to claim 1 or 19, or a combination thereof; and causing expression of the first polypeptide, the second polypeptide, or a combination thereof. 23. The method according to claim 22, where the expression vector encoding the first polypeptide and the expression vector encoding the second polypeptide are co-transfected into cells, or the expression vector encoding the first polypeptide and the expression vector encoding the second polypeptide are respectively transformed at least two cell type. 24. The method according to claim 22, comprising obtaining a protein complex based on the first polypeptide, the second polypeptide or the first polypeptide and the second polypeptide from cells or cell culture medium. 25. The method of claim 22, wherein the first polypeptide and/or the second polypeptide further comprise one selected from the group consisting of an antigen binding fragment (Fab), a single chain variable fragment (scFv), a membrane receptor extracellular domain, an agonist, an antagonist, a ligand , decoy receptor, cytokine, coagulation factor and affinity tag.