WO2023068818A1 - 두개의 fc 도메인을 포함하는 항원 결합 단백질 및 이의 용도 - Google Patents
두개의 fc 도메인을 포함하는 항원 결합 단백질 및 이의 용도 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
Definitions
- the present invention relates to a novel antibody construct having an antigen binding site that specifically binds to a cancer surface antigen and two Fc domains.
- Antibody-based therapeutics and Fc fusion proteins are a group of clinically important drugs for patients with cancer, immune disorders, infectious and inflammatory diseases.
- ADCC Antibody-Dependent Cell-mediated Cytotoxicity
- ADCP Antibody-Dependent Cellular Phagocytosis
- CDC Complement-Dependent Cytotoxicity
- the present inventors studied to improve the function of the antibody and solve the problems of the existing antibody structure designed to include multiple Fc domains in series as described above.
- natural human immunoglobulin G IgG
- Compared to natural human antibodies despite having a similar molecular weight (about 150 kDa), improved novel antibody constructs were developed that allow up to four times the Fc domain to be present on cell surface antigens.
- One aspect of the present invention comprises an antigen-binding site, a first Fc domain linked to the first linking position of the antigen-binding site or a variant thereof, and a second Fc domain linked to the second linking position of the antigen-binding site or a variant thereof. It is to provide a fusion protein comprising
- Another aspect of the present invention is two antigen-binding regions connected in series (tandem two antigen-binding regions), a first Fc domain linked to the first linking position of the tandem 2 antigen-binding region or a variant thereof, and the tandem 2 It is to provide a fusion protein comprising a second Fc domain linked to a second linking position of an antigen binding site or a variant thereof.
- each of the two antigen-binding sites constituting the tandem 2 antigen-binding site binds to different epitopes of the same antigen or binds to different antigens, respectively CDR sequences or variable regions (comprising) Or it may be a sequence consisting of a variable region (consisting of).
- the first Fc domain and the second Fc domain may or may not be linked to each other through a covalent bond, a non-covalent bond, or a linker. there is. In a preferred embodiment, the first Fc domain and the second Fc domain are not linked to each other.
- the Fc domain may be a wild-type immunoglobulin Fc domain, and modulates the Fc ⁇ receptor (Fc ⁇ R) reactivity or ADCC of the Fc domain, or multimer formation of undesirable Fc domains. Modifications to minimize , such as amino acid substitutions, may be included.
- the Fc domain includes the CH2 and CH3 regions, may include the CH4 region and/or the hinge region, and should be interpreted as including Fc domain fragments representing the functions of the Fc domain.
- binding between the antigen-binding site and the Fc domain or variant may be direct binding or binding through a linker.
- binding of the antigen-binding site to the Fc domain or variant may be binding between the N-terminus and the C-terminus, the N-terminus and the N-terminus, or the C-terminus and the C-terminus of each peptide molecule. .
- 13c shows the result of sensorgram analysis of binding of H01Fv4, a purified Fv-(Fc)2 construct, to human HER2.
- 17a is a result of measuring the amount of Fc domain present on the surface of NCI-N87 gastric cancer cell line upon treatment with 50 nM HER2 antibody.
- 17b shows the results of measuring the amount of Fc domain present on the surface of BT474 breast cancer cell line upon treatment with 50 nM HER2 antibody.
- 17c shows the results of measuring the amount of Fc domain present on the surface of SK-OV3 ovarian cancer cell line upon treatment with 50 nM HER2 antibody.
- 17e is a result of measuring the amount of Fc domain present on the surface of SNU-5 gastric cancer cell line upon treatment with 50 nM HER2 antibody.
- 18a is a result of measuring the amount of Fc domain present on the surface of NCI-N87 gastric cancer cell line upon treatment with various concentrations of HER2 antibody.
- 19a is a schematic structural diagram of H01DE4 in which S239D and I332E mutations were introduced using H01 as a template.
- 19B is a schematic structural diagram of P01DE4 in which S239D and I332E mutations were introduced using P01 as a template.
- 20a to 20d are sensorgrams showing the binding characteristics and affinity of H01, H01DE4, P01, and P01DE4 to human HER2.
- 21a to 21h are sensorgrams showing the binding characteristics and affinity of H01, P01, H01DE4, P01DE4, Human IgG1, Trastuzumab, Pertuzumab, and Margetuximab to Fc ⁇ receptor 1.
- 23a to 23h are sensorgrams showing binding characteristics and affinity of H01, P01, H01DE4, P01DE4, Human IgG1, Trastuzumab, Pertuzumab, and Margetuximab to Fc ⁇ receptor 3A (176V isoform).
- 25 is a structural schematic diagram of HP501, an improved HER2 biparatopic antibody.
- 26 is a structural schematic diagram of HER2 biparatopic modified antibodies, HP501 to HP516.
- 28a is a sensorgram of affinity analysis of HP503 to HER2 protein using Bio-Layer Interferometry analysis.
- 28B is a sensorgram of affinity analysis of HP507 for HER2 protein using Bio-Layer Interferometry.
- 28c is a sensorgram of affinity analysis of HP511 to HER2 protein using Bio-Layer Interferometry.
- 29a is a graph showing the results of analyzing the binding characteristics and affinity of HP503 to Fc ⁇ receptor 1, Fc ⁇ receptor 2A (131R isoform), and Fc ⁇ receptor 3A (176V isoform).
- 29B is a graph showing the results of analyzing the binding characteristics and affinity of HP507 to Fc ⁇ receptor 1, Fc ⁇ receptor 2A (131R isoform), and Fc ⁇ receptor 3A (176V isoform).
- 30A is a graph showing the results of analyzing the binding characteristics and affinity of HP503, HP507, HP511 and HP515 to the neonatal Fc receptor (FcRn).
- 30B is a graph showing the results of analyzing the binding characteristics and affinity of human IgG1, Trastuzumab, Pertuzumab, and Margetuximab to the neonatal Fc receptor (FcRn).
- 31a shows the results of analyzing the CDC effects of each HER2 antibody in the BT474 breast cancer cell line.
- 32a is a result of analyzing the ADCC effect by the HER2 antibody in the NCI-N87 gastric cancer cell line.
- Figure 32b is the result of analyzing the ADCC effect by HER2 antibody in MDA-MB-453 breast cancer cell line.
- Figure 32c is the result of analyzing the ADCC effect by HER2 antibody in SNU-601 gastric cancer cell line.
- Figure 32d is the result of analyzing the ADCC effect by HER2 antibody in SNU-5 gastric cancer cell line.
- Figure 33a shows the results of anticancer activity analysis in SNU-5 gastric cancer cell line xenograft mouse model (C.B-17 SCID).
- 33b shows the results of anticancer activity analysis in SNU-5 gastric cancer cell line xenograft mouse model (BALB/c-nu).
- 36 is a vector capable of expressing human HER2 protein in mammalian cells.
- FIG. 39 compares the relative human HER2 expression level of CT26-HER2 cell line (Clone name: #2-60) compared to human cancer cell lines and confirms that H01 binds a larger amount of Fc domain to the surface of CT26-HER2 cells compared to Trastuzumab. This is the result.
- 40 is an analysis result of anticancer efficacy in an allogeneic mouse model transplanted with CT26-HER2.
- 41a is a schematic diagram of a monovalent engineered mAb according to one embodiment.
- 41B is a schematic diagram of a Biparatopic engineered mAb according to one embodiment.
- FIG. 42 is a sensorgram result of analyzing the target-binding ability of a fusion protein, which is an embodiment.
- FIG. 42a is a sensorgram result of analyzing the binding ability of GPM01, a monovalent engineered mAb targeting GPC-3, to human GPC-3.
- Figure 42b is a sensorgram result of analyzing the binding ability of GPM02, a monovalent engineered mAb targeting GPC-3, to human GPC-3.
- 42c is a sensorgram result of analyzing the binding ability of GPM04, a monovalent engineered mAb targeting GPC-3, to human GPC-3.
- 42D is a sensorgram result of analyzing the binding ability of GPB01, a biparatopic engineered mAb targeting GPC-3, to human GPC-3.
- 42E is a sensorgram result of analyzing the binding ability of GPB03, a biparatopic engineered mAb targeting GPC-3, to human GPC-3.
- 42f is a sensorgram result of analyzing the binding ability of GPB04, a biparatopic engineered mAb targeting GPC-3, to human GPC-3.
- 42g is a sensorgram result of analyzing the binding ability of GPB06, a biparatopic engineered mAb targeting GPC-3, to human GPC-3.
- MEM01 and MEM06 are monovalent engineered mAbs targeting MET, to human MET.
- 49 is a sensorgram result of analyzing the binding ability of monovalent engineered mAbs targeting EGFR to human EGFR.
- Figure 50 is a sensorgram for analyzing the binding ability to human CD33 of monovalent engineered mAbs 33-1, 33-2, 33-3 and biparatopic engineered mAbs 33-4, 33-5, 33-6, 33-7 targeting CD33 This is the result.
- FIG. 52 is a sensorgram result of analyzing the target-binding ability of a fusion protein, which is an embodiment.
- FIG. 52a is a sensorgram result of analyzing the binding ability of T01, a monovalent engineered mAb targeting TROP2, to human TROP2.
- 52B is a sensorgram result of analyzing the binding ability of MSM01, a monovalent engineered mAb targeting mesothelin, to human mesothelin.
- 52c is a sensorgram result of analyzing the binding ability of LIMO1, a monovalent engineered mAb targeting LIV-1, to human LIV-1.
- fusion protein having two Fc or "antibody having two Fc” refers to a fusion protein in which two Fc domains are independently linked to two polypeptide chains constituting an antigen binding site. do.
- the two polypeptide chains constituting the antigen binding site may differ from one another.
- one of the two polypeptide chains constituting the antigen binding site may be a sequence comprising or consisting of the light chain CDR sequence or light chain variable region of an antibody, or may be a scFv, and the other may be an antibody heavy chain CDR sequence or It may be a sequence comprising or consisting of a heavy chain variable region or a scFv.
- the fusion protein having the two Fc regions may include a “humanized” form of a non-human antibody, which is a chimeric antibody containing human immunoglobulin including natural CDRs.
- the fusion protein may include a "fully human antibody” or a part of a "human antibody”.
- the multi-specific fusion protein or antigen binding domain may be a "monoclonal antibody” or part thereof.
- the term "heavy chain” refers to a polypeptide chain between about 50 kDa and about 70 kDa.
- the N-terminal portion includes a variable region of about 120 to 130 or more amino acids
- the C-terminal portion includes a constant region.
- a constant region can be one of five types: alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ), and mu ( ⁇ ).
- ⁇ , ⁇ and ⁇ contain about 450 amino acids and ⁇ and ⁇ contain about 550 amino acids.
- the term "light chain” refers to a polypeptide chain of about 25 kDa.
- the N-terminal portion includes a variable region of about 100 to about 110 or more amino acids
- the C-terminal portion includes a constant region.
- the constant region of the light chain is referred to as "CL”.
- Heavy chain C domains (CH domains) are numbered from N-terminus to C-terminus (eg, CH 1 , CH 2 , CH 3 , etc.).
- CL and CH 1 regions of any of these antibody classes may be used in the present disclosure.
- CL and CH 1 regions provided herein are of the IgG type (eg, IgG1).
- Fc refers to the C-terminal region of an immunoglobulin heavy chain, including native Fc regions, recombinant Fc regions and variant Fc regions.
- Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the hinge present at the N-terminal of these domains.
- Fc may include a J chain.
- Fc contains the immunoglobulin domains Cy2 (CH2) and Cy3 (CH3) and a hinge between Cy1 and Cy2.
- a variant Fc region has at least one amino acid substitution relative to a native sequence Fc region; For example, from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acids may be substituted
- the variant Fc region is at least about 80% homologous to the native sequence Fc region, and at least about 90% homology, or at least about 95% homology.
- single-chain Fv or “scFv” refers to an antibody fragment comprising the VH and VL domains of an antibody to exist within a single polypeptide chain.
- variable region refers to any of the VL (including Vkappa (VK) and Vlamda (VL)) and/or VH genes constituting the light chain (including kappa and lambda) and heavy chain immunoglobulin loci, respectively.
- VL or VH means a region of an antibody comprising one or more immunoglobulin domains encoded by one.
- the light or heavy chain variable region (VL or VH) consists of "framework" or "FR" regions comprising three hypervariable regions called “complementarity determining regions” or "CDRs".
- the term "antigen” is a structure capable of selectively binding to an antibody.
- a target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. Specifically, the antigen is a polypeptide and may be a protein present on or within a cell.
- epitope refers to an antigenic determinant and is a region on an antigen to which an antibody or polypeptide binds. Protein epitopes can include amino acid residues directly involved in binding as well as amino acid residues that are effectively blocked by specific antigen-binding antibodies or peptides. It is the simplest form or smallest structural region of a complex antigenic molecule capable of binding to an antibody or receptor. Epitopes can be linear or conformational/conformational.
- vector refers to a material for transporting or expressing a nucleic acid sequence including a nucleic acid sequence encoding a multi-specific fusion protein (eg, antibody) described herein.
- vectors include expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes.
- One aspect of the present invention provides an antibody comprising a plurality of Fc domains, characterized in that the ratio of the antigen binding site and the Fc domain is 1:2 or 2:2.
- the antibody may be a fusion protein comprising an antigen binding site, a first Fc domain or variant thereof, and a second Fc domain or variant thereof.
- the antigen binding site may be composed of two different polypeptide chains.
- each polypeptide may be linked to a first Fc domain or variant thereof and a second Fc domain or variant thereof.
- the two Fc domains may be fusion proteins linked to the C-terminus of the CH1 region of the heavy chain and the C-terminus of the constant region of the light chain, respectively.
- the Fc domain and Fab can be linked through a peptide linker.
- the two Fc domains may be a fusion protein linked to the C-terminus of the heavy chain variable region and the C-terminus of the light chain variable region, respectively.
- the Fc domain and Fv may be linked through a peptide linker.
- This novel antibody construct has a molecular weight similar to that of human IgG.
- the fusion protein may have an antigen binding affinity equal to that of a human IgG-based antibody.
- the Fc domain may be present on the cell surface antigen up to 4 times that of natural human antibodies. Due to these characteristics, the fusion protein can increase Fc ⁇ receptor affinity and effector functions compared to wild-type antibodies.
- Each Fc domain bound to the fusion protein may have a similar level of Fc receptor (Fc ⁇ receptor and FcRn) binding affinity to that of the Fc domain of an IgG-based antibody, but due to the avidity effect, the fusion
- the protein's Fc receptor (Fc ⁇ receptor and FcRn) apparent affinity may have a significantly increased value compared to human IgG antibody.
- the fusion protein has a similar level of thermal stability to IgG-based antibodies.
- the fusion protein is composed of (a) a first polypeptide comprising at least one complementarity-determining region (CDR) sequence and a second polypeptide comprising at least one complementarity-determining region (CDR) sequence, wherein The first polypeptide and the second polypeptide form a dimer, an antigen-binding site capable of specifically binding to a target antigen, (b) a dimer composed of two polypeptide sequences, one of which is a polypeptide sequence A first Fc domain or a variant thereof linked to the first polypeptide of the antigen-binding site, and (c) a dimer of two polypeptide sequences, wherein one polypeptide sequence binds to the second polypeptide of the antigen-binding site. It may be a fusion protein comprising a second Fc domain or a variant thereof.
- the first polypeptide of the antigen-binding portion may include CDR1, CDR2, and CDR3 of the antibody heavy chain
- the second polypeptide of the antigen-binding portion may include CDR1, CDR2, and CDR3 of the antibody light chain
- the first polypeptide of the antigen-binding site may further include a CH1 region of an antibody heavy chain
- the second polypeptide of the antigen-binding site may further include a constant region of an antibody light chain.
- the antigen binding site may specifically bind to a protein expressed on the cell surface.
- the antigen binding site may specifically bind to a cancer antigen.
- the antigen binding site is PD-L1, EGFR, EGFRvIII, BCMA, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, c-Met (MET), DLL3, DR4, DR5 , GD2, Nectin-4, RANKL, SLAMF7, Trop-2, LIV-1, Claudin 18.2, IL13 ⁇ 2, CD3, HER2, HER3, FGFR2, FGFR3, GPC3, ROR1, Fol ⁇ , CD20, CD19, CTLA-4, VEGFR, NCAM1, ICAM-1, ICAM-2, CEACAM5, CEACAM6, Carcinoembryonic antigen (CEA), CA-125, Alphafetoprotein (AFP), MUC-1, MUC-16, PSMA, PSCA, Epithelial tumor antigen (ETA), Melanoma- associated antigen (MAGE), Immature laminin receptor, TAG-72, HPV E6/E7, BING-4, Calcium-
- the second antigen-binding site may also specifically bind to any one antigen selected from the above group.
- the antigen to which the first antigen-binding site binds and the antigen to which the second antigen-binding site binds may be different.
- the first antigen-binding site may include a sequence that specifically binds to HER2 and the second antigen-binding site may include a sequence that specifically binds to EGFR.
- the first antigen-binding site may include a sequence that specifically binds to one epitope of an antigen and the second antigen-binding site may include a sequence that specifically binds to a different epitope of the same antigen.
- the antigen binding site may include a variable region that specifically binds to the antigen.
- the variable region is Cetuximab, Panitumumab, Necitumumab, Imgatuzumab, Depatuxizumab, Losatuxizumab, Etevritamab, AMG-595, Atezolizumab, Avelumab, Durvalumab, Trastuzumab, Pertuzumab, Onartuzumab, Emibetuzumab, Telisotuzumab, Datopotamab, Tarbalatamab, Belantamapituzumab, and Rovalamapituzumab.
- Ladiratuzumab, Codrituzumab, Aprutumab, Bemarituzumab, Vofatamab, Ramucirumab, Rituximab, Obinutuzumab, Daratumumab, and 1C1 may include a heavy chain variable region and a light chain variable region of any one antibody, but are limited thereto It doesn't work.
- Cetuximab may include a heavy chain variable region including H-CDR1 represented by SEQ ID NO: 175, H-CDR2 represented by SEQ ID NO: 176, and H-CDR3 represented by SEQ ID NO: 177, and SEQ ID NO: 178 It may include a light chain variable region including L-CDR1 represented by SEQ ID NO: 179, L-CDR2 represented by SEQ ID NO: 179, and L-CDR3 represented by SEQ ID NO: 180.
- Another example may include a heavy chain variable region including H-CDR1 represented by SEQ ID NO: 181, H-CDR2 represented by SEQ ID NO: 182, and H-CDR3 represented by SEQ ID NO: 183 of Panitumumab, It may include a light chain variable region including L-CDR1 represented by 184, L-CDR2 represented by SEQ ID NO: 185, and L-CDR3 represented by SEQ ID NO: 186.
- Necitumumab may include a heavy chain variable region including H-CDR1 represented by SEQ ID NO: 187, H-CDR2 represented by SEQ ID NO: 188, and H-CDR3 represented by SEQ ID NO: 189, It may include a light chain variable region including L-CDR1 represented by 190, L-CDR2 represented by SEQ ID NO: 191, and L-CDR3 represented by SEQ ID NO: 192.
- H-CDR1 represented by SEQ ID NO: 193, H-CDR2 represented by SEQ ID NO: 194, and H-CDR3 represented by SEQ ID NO: 195 of Imgatuzumab, represented by SEQ ID NO: 196
- It may include a light chain variable region including L-CDR1 represented by SEQ ID NO: 197, L-CDR2 represented by SEQ ID NO: 197, and L-CDR3 represented by SEQ ID NO: 198.
- H-CDR1 represented by SEQ ID NO: 199
- H-CDR2 represented by SEQ ID NO: 200
- H-CDR3 represented by SEQ ID NO: 201 of Depatuxizumab
- SEQ ID NO: 202 It may include a light chain variable region including L-CDR1 represented by SEQ ID NO: 203, L-CDR2 represented by SEQ ID NO: 203, and L-CDR3 represented by SEQ ID NO: 204.
- H-CDR1 represented by SEQ ID NO: 199
- H-CDR2 represented by SEQ ID NO: 205
- H-CDR3 represented by SEQ ID NO: 206 of Losatuxizumab
- SEQ ID NO: 202 It may include a light chain variable region including L-CDR1 represented by SEQ ID NO: 203, L-CDR2 represented by SEQ ID NO: 203, and L-CDR3 represented by SEQ ID NO: 204.
- an antigen binding site that specifically binds to EGFRvIII may be included.
- it may include a heavy chain variable region including H-CDR1 represented by SEQ ID NO: 207, H-CDR2 represented by SEQ ID NO: 208, and H-CDR3 represented by SEQ ID NO: 209 of Etevritamab, and SEQ ID NO: 210
- It may include a light chain variable region including L-CDR1 represented by SEQ ID NO: 211, L-CDR2 represented by SEQ ID NO: 211, and L-CDR3 represented by SEQ ID NO: 212.
- H-CDR1 represented by SEQ ID NO: 213 of AMG-595
- H-CDR2 represented by SEQ ID NO: 214
- H-CDR3 represented by SEQ ID NO: 215, and SEQ ID NO: 210
- L-CDR1 represented by SEQ ID NO: 216
- L-CDR2 represented by SEQ ID NO: 216
- L-CDR3 represented by SEQ ID NO: 217.
- an antigen binding site that specifically binds to PD-L1 may be included.
- atezolizumab may include a heavy chain variable region including H-CDR1 represented by SEQ ID NO: 218, H-CDR2 represented by SEQ ID NO: 219, and H-CDR3 represented by SEQ ID NO: 220, and SEQ ID NO: 221 It may include a light chain variable region including L-CDR1 represented by SEQ ID NO: 222, L-CDR2 represented by SEQ ID NO: 222, and L-CDR3 represented by SEQ ID NO: 223.
- H-CDR1 represented by SEQ ID NO: 224
- H-CDR2 represented by SEQ ID NO: 225
- H-CDR3 represented by SEQ ID NO: 226 of Avelumab, represented by SEQ ID NO: 227
- L-CDR1 represented by SEQ ID NO: 228, L-CDR2 represented by SEQ ID NO: 228, and L-CDR3 represented by SEQ ID NO: 229.
- an antigen binding site that specifically binds to HER2 may be included.
- Trastuzumab may include a heavy chain variable region including H-CDR1 represented by SEQ ID NO: 21, H-CDR2 represented by SEQ ID NO: 22, and H-CDR3 represented by SEQ ID NO: 23, and SEQ ID NO: 24 It may include a light chain variable region including L-CDR1 represented by SEQ ID NO: 25, L-CDR2 represented by SEQ ID NO: 25, and L-CDR3 represented by SEQ ID NO: 26.
- an antigen binding site that specifically binds to the CD20 may be included.
- Rituximab may include a heavy chain variable region including H-CDR1 represented by SEQ ID NO: 314, H-CDR2 represented by SEQ ID NO: 315, and H-CDR3 represented by SEQ ID NO: 316, and SEQ ID NO: 317 It may include a light chain variable region including L-CDR1 represented by SEQ ID NO: 318, L-CDR2 represented by SEQ ID NO: 318, and L-CDR3 represented by SEQ ID NO: 319.
- HER-2/neu human epidermal growth factor receptor 2
- PI3K/AkT PI3K/AkT
- Her2/neu target The anticancer agent may be Trastuzumab or Pertuzumab, but is not limited thereto.
- FGFR Fibroblast growth factor receptor
- FGF fibroblast growth factor
- the FGFR gene is frequently mutated, and these variants are commonly observed in breast cancer, uterine cancer, ovarian cancer, cervical cancer, and the like.
- Four FGFR genes are made of seven signaling receptors, among which FGFR2 and FGFR3 are highly cancer-specific antigens.
- An antibody targeting FGFR2 or FGFR3 may be Aprutumab, Bemarituzumab, or Vofatamab, but is not limited thereto.
- EphA2 (EPH receptor A2): It is overexpressed in cancer cells and has a great effect on the growth and metastasis of cancer cells, and an antibody targeting this may be 1C1, but is not limited thereto.
- VH region comprising the amino acid sequences of SEQ ID NO: 218 (VH-CDR1), SEQ ID NO: 219 (VH-CDR2) and SEQ ID NO: 220 (VH-CDR3) (SEQ ID NO: 350), and SEQ ID NO: 221 (VL-CDR1 ), the VL region (SEQ ID NO: 351) comprising the amino acid sequences of SEQ ID NO: 222 (VL-CDR2) and SEQ ID NO: 223 (VL-CDR3);
- VH region comprising the amino acid sequences of SEQ ID NO: 236 (VH-CDR1), SEQ ID NO: 237 (VH-CDR2) and SEQ ID NO: 238 (VH-CDR3) (SEQ ID NO: 358), and SEQ ID NO: 239 (VL-CDR1)
- VL region SEQ ID NO: 359 comprising the amino acid sequences of SEQ ID NO: 240 (VL-CDR2) and SEQ ID NO: 241 (VL-CDR3)
- VH region comprising the amino acid sequences of SEQ ID NO: 266 (VH-CDR1), SEQ ID NO: 267 (VH-CDR2) and SEQ ID NO: 268 (VH-CDR3) (SEQ ID NO: 368), and SEQ ID NO: 269 (VL-CDR1) ), the VL region (SEQ ID NO: 369) comprising the amino acid sequences of SEQ ID NO: 270 (VL-CDR2) and SEQ ID NO: 271 (VL-CDR3);
- VH region comprising the amino acid sequences of SEQ ID NO: 99 (VH-CDR1), SEQ ID NO: 100 (VH-CDR2) and SEQ ID NO: 101 (VH-CDR3) (SEQ ID NO: 87), and SEQ ID NO: 102 (VL-CDR1)
- VL region comprising the amino acid sequences of SEQ ID NO: 103 (VL-CDR2) and SEQ ID NO: 104 (VL-CDR3)
- VH region comprising the amino acid sequences of SEQ ID NO: 302 (VH-CDR1), SEQ ID NO: 303 (VH-CDR2) and SEQ ID NO: 304 (VH-CDR3) (SEQ ID NO: 380), and SEQ ID NO: 305 (VL-CDR1)
- VL region SEQ ID NO: 381 comprising the amino acid sequences of SEQ ID NO: 306 (VL-CDR2) and SEQ ID NO: 307 (VL-CDR3)
- VEGFR2 VEGFR2
- VH region comprising the amino acid sequences of SEQ ID NO: 308 (VH-CDR1), SEQ ID NO: 309 (VH-CDR2) and SEQ ID NO: 310 (VH-CDR3) (SEQ ID NO: 382), and SEQ ID NO: 311 (VL-CDR1) ), the VL region (SEQ ID NO: 383) comprising the amino acid sequences of SEQ ID NO: 312 (VL-CDR2) and SEQ ID NO: 313 (VL-CDR3);
- VH region comprising the amino acid sequences of SEQ ID NO: 314 (VH-CDR1), SEQ ID NO: 315 (VH-CDR2) and SEQ ID NO: 316 (VH-CDR3) (SEQ ID NO: 384), and SEQ ID NO: 317 (VL-CDR1) ), the VL region (SEQ ID NO: 385) comprising the amino acid sequences of SEQ ID NO: 318 (VL-CDR2) and SEQ ID NO: 319 (VL-CDR3);
- VH region comprising the amino acid sequences of SEQ ID NO: 320 (VH-CDR1), SEQ ID NO: 321 (VH-CDR2) and SEQ ID NO: 322 (VH-CDR3) (SEQ ID NO: 386), and SEQ ID NO: 323 (VL-CDR1) ), the VL region (SEQ ID NO: 387) comprising the amino acid sequences of SEQ ID NO: 324 (VL-CDR2) and SEQ ID NO: 325 (VL-CDR3);
- VH region comprising the amino acid sequences of SEQ ID NO: 326 (VH-CDR1), SEQ ID NO: 327 (VH-CDR2) and SEQ ID NO: 328 (VH-CDR3) (SEQ ID NO: 388), and SEQ ID NO: 329 (VL-CDR1) ), the VL region (SEQ ID NO: 389) comprising the amino acid sequences of SEQ ID NO: 330 (VL-CDR2) and SEQ ID NO: 331 (VL-CDR3);
- VH region comprising the amino acid sequences of SEQ ID NO: 157 (VH-CDR1), SEQ ID NO: 158 (VH-CDR2) and SEQ ID NO: 159 (VH-CDR3) (SEQ ID NO: 143), and SEQ ID NO: 160 (VL-CDR1)
- VL region SEQ ID NO: 145) comprising the amino acid sequences of SEQ ID NO: 161 (VL-CDR2) and SEQ ID NO: 162 (VL-CDR3).
- Table 2 shows nucleotide sequences and polypeptide sequences of exemplary signal sequences for efficiently expressing fusion proteins according to various embodiments.
- SEQ ID NO: 333 may be used as a signal sequence, but is not limited thereto.
- variable region polypeptide sequences of anticancer antibodies described as antigen binding sites of various fusion proteins described herein Fusion proteins according to exemplary embodiments may comprise (comprising) these variable region polypeptides or consist of these variable region polypeptides (consists of).
- Table 4 shows the nucleotide sequences of the variable regions of anti-cancer antibodies described herein as antigen-binding sites of various fusion proteins.
- each of the above-described first Fc domain and the second Fc domain may be an Fc region of an immunoglobulin.
- the Fc region of the immunoglobulin may be a wild-type Fc domain as well as an Fc domain variant.
- the Fc region may be an IgG, IgA, IgE, IgD or IgM Fc region.
- Fc domain variant refers to a glycosylation pattern different from that of the wild-type Fc domain, increased sugar chains compared to the wild-type Fc domain, decreased sugar chains compared to the wild-type Fc domain, or deglycosylated sugar chains.
- the Fc domain or variant may have sialic acid, fucosylation, and glycosylation of numbers adjusted through culture conditions or genetic manipulation of the host.
- the sugar chain of the Fc domain of immunoglobulin can be modified by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms.
- the Fc domain variant may be a mixed form of an Fc region of immunoglobulin IgG, IgA, IgE, IgD or IgM.
- the Fc domain variant may be a form in which some amino acids of the Fc domain are substituted with other amino acids.
- Amino acid introduced by the above substitution and/or addition is lysine (K), alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q ), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T ), it may be any one selected from the group consisting of tryptophan (W), tyrosine (Y) and valine (V).
- Fc region may be a form in which amino acids 239 and/or 332 of the CH2 region are substituted with other amino acids (see Kabat numbering system).
- S239 may be substituted with an amino acid other than S, and specifically, may be substituted with S239D.
- I332 may be substituted with an amino acid other than I, and specifically, may be substituted with I332E.
- the Fc region may include a knob structure or a hole structure.
- the term "knob-into-hole” refers to antibodies that specifically bind to different regions, such as bispecific antibodies, multispecific antibodies, or heterodimeric antibodies. It is a design strategy for manufacturing. Generally, this technique involves placing a knob on the interface of a first polypeptide (e.g., the first CH3 domain of a first antibody heavy chain) and a knob on the interface of a second polypeptide (e.g., the second CH3 domain of a second antibody heavy chain). This may include introducing a corresponding hole, such that a knob is positioned within the hole to promote heterodimer formation and hinder homodimer formation.
- a first polypeptide e.g., the first CH3 domain of a first antibody heavy chain
- a knob on the interface of a second polypeptide e.g., the second CH3 domain of a second antibody heavy chain
- a 'knob' is constructed by replacing small amino acid side chains from the interface of the first polypeptide (eg the first CH3 domain of the first antibody heavy chain) with larger side chains (eg arginine, phenylalanine, tyrosine or tryptophan).
- Complementary 'holes' of the same or similar size in the knob may be formed at the interface of the second polypeptide (eg, the second CH3 domain of the second antibody heavy chain) by replacing the large amino acid side chains with smaller side chains (eg, alanine, serine, valine, or threonine).
- the knobs and holes can be created by altering the nucleic acid encoding the polypeptide, eg, by site-directed mutagenesis or by peptide synthesis.
- WO2014084607A1 is, for example, a CH3 domain (a-1) binding of tryptophan (W) substituted at Lys409 of one CH3 domain and valine (V) substituted at Asp399 of another CH3 domain and threonine (T) substituted at Phe405.
- W tryptophan
- V valine
- T threonine
- (a-2) a combination of serine (S) substituted at Tyr349 of one CH3 domain and tryptophan (W) substituted at Glu357 of another CH3 domain, and in addition (b-1) Lys360 of one CH3 domain.
- the positions of the amino acid residues are according to the EU index.
- WO2018059502A1 describes, for example, mutations in the Fc domain comprising one or more mutations selected from each of the following a) - e): a) L351G, L351Y, L351V, L351P, L351D, L351E, L351K, or L351W; b) T366L, T366P, T366W, or T366V;c) D399C, D399N, D399I, D399G, D399R, D399T, or D399Y, 0) Y407P, Y407F, Y407T, or Y407H; and e) K409C , K409P, K409S, K409F, K409V, K409Q, or K409R.
- the positions of the amino acid residues are according to the EU index.
- n and m are each independently 0 or 1
- the X includes a heavy chain variable region or a light chain variable region of an antibody that specifically binds to an antigen
- Y includes a light chain variable region or a heavy chain variable region of an antibody that specifically binds to an antigen
- the X and Y combine with each other to form an antigen-binding site (a) that specifically binds to an antigen
- polypeptides of (I), (II), (III) and (IV) may combine to form one antigen-binding site and two Fc domains.
- the X is the first polypeptide sequence of the antigen-binding site, and is a sequence comprising the heavy chain CDR1, CDR2, or CDR3 sequence of an antibody that specifically binds to the first antigen, or a sequence that specifically binds to the first antigen. It includes a heavy chain variable region of an antibody; Y is the second polypeptide sequence of the antigen-binding site, and is a sequence comprising the light chain CDR1, CDR2, or CDR3 sequences of an antibody that specifically binds to the first antigen, or a light chain of an antibody that specifically binds to the first antigen. contains a variable region; The X and Y combine to form the antigen-binding site (a) that specifically binds to an antigen.
- the CH3 region may be mutated so that interactions between A and B and C and D are minimized and heterodimeric Fc formation between A and C and B and D is promoted.
- the Fc domain monomer comprises a knob structure or a hole structure that promotes the formation of an Fc heterodimer (heterodimeric Fc);
- the Fc domain monomer may include a variant promoting heterodimer formation by an electrostatic steering mechanism.
- X of Structural Formula (I) may additionally include a heavy chain CH1 region, and/or Y of Structural Formula (II) may additionally include a light chain constant region.
- the fusion protein may include polypeptide chains represented by the following structural formulas (I'), (II'), (III) and (IV):
- N' is the N-terminus of the polypeptide chain
- C' is the C-terminus of the polypeptide chain
- A, B, C and D are monomeric polypeptide sequences of an Fc domain comprising CH2 and CH3 regions of an immunoglobulin, optionally further comprising CH4 and/or a hinge sequence, wherein A is combined with one of C or D dimerize together to form the first Fc domain (b), and B dimerize with the remaining one of C or D to form the second Fc domain (c);
- n, m, p and q are each independently 0 or 1
- the VD1 is composed of an antibody heavy or light chain variable region or an antibody heavy or light chain CDR1, CDR2, and CDR3 that specifically binds to an antigen;
- the VD2 is composed of a light chain or heavy chain variable region of an antibody that specifically binds to an antigen or an antibody heavy or light chain CDR1, CDR2, and CDR3;
- the X includes an antibody heavy or light chain variable region or an antibody heavy or light chain CDR1, CDR2, and CDR3 that specifically bind to an antigen;
- X and Y combine to form a first antibody variable region that specifically binds to the first antigen
- the CH3 region may be mutated so that interactions between A and B and C and D are minimized and heterodimeric Fc formation between A and C and B and D is promoted.
- the Fc domain monomer comprises a knob structure or a hole structure that promotes the formation of an Fc heterodimer (heterodimeric Fc);
- the Fc domain monomer may include a variant promoting heterodimer formation by an electrostatic steering mechanism.
- the heavy chain variable region may additionally include a heavy chain CH1 region.
- the light chain variable region may additionally include a light chain constant region.
- a hinge is an immunoglobulin-derived hinge region.
- the antibody hinge region is an IgG hinge region.
- An IgG hinge region provided herein can be selected from, for example, antibody hinge regions of various IgG subtypes. The table below lists exemplary IgG subtypes with core hinge sequences that can be included in the flexible peptide regions provided herein.
- at least one Cys may exist in the hinge. Specifically, one, two, or three Cys may exist in the hinge.
- the hinge can be modified to delete disulfide bonds or introduce additional disulfide bonds.
- each of the linkers L1 and L2 may include 1 to about 70 amino acids.
- the L1 and L2 may each include about 5 to about 60, about 10 to about 50, about 15 to about 40, and about 20 to about 30 amino acids.
- L1 and L2 are each 1-70 amino acid residues, 2-60 amino acid residues, 2-50 amino acid residues, 2-40 amino acid residues, 2-30 amino acid residues, 3- 50 amino acid residues, 3-40 amino acid residues, 3-30 amino acid residues, 2-28 amino acid residues, 2-26 amino acid residues, 2-24 amino acid residues, 2-22 amino acid residues, 2-20 amino acid residues, 2-18 amino acids residues, 2-16 amino acid residues, 2-14 amino acid residues, 2-12 amino acid residues, 2-10 amino acid residues.
- the L1 and L2 may include the amino acid sequence of (G4S)o (where o is an integer from 1 to 5) in Table 6 below, but is not limited thereto.
- L1 and L2 may have different amino acid sequences.
- L1 and L2 may include at least one Cys.
- a disulfide bond may be formed through Cys present in L1 and L2.
- each of L3 and L4 may include 1 to about 30 amino acids.
- the L3 and L4 may each include about 5 to about 25, about 10 to about 20, and about 15 amino acids.
- L3 and L4 are each 2-30 amino acid residues, 2-25 amino acid residues, 2-20 amino acid residues, 2-15 amino acid residues, 3-30 amino acid residues, 2-28 amino acid residues, 2-26 amino acid residues, 2-24 amino acid residues, 2-22 amino acid residues, 2-20 amino acid residues, 2-18 amino acid residues, 2-16 amino acid residues, 2-14 amino acid residues, 2-12 amino acid residues, 2- It can be a peptide consisting of 10 amino acid residues.
- the L3 and L4 may include the amino acid sequence of (G4S) o (where o is an integer from 1 to 5) in Table 6, but is not limited thereto. Also, L3 and L4 may have different amino acid sequences.
- Fusion protein containing one antigen binding site and two Fc
- the fusion protein includes polypeptides of structural formulas (I), (II), (III) and (IV), wherein X is a heavy chain variable region and additionally includes CH1, and Y is As a light chain variable region, it includes a light chain constant region. In addition, it binds to the CH1 structure and Cys in the light chain variable region to form a Fab structure.
- n is 0, CH1 is directly bonded to the hinge, m is 1, and L2 includes a peptide linker.
- a and C combine to form a first Fc domain
- B and D combine to form a second Fc domain.
- a hole structure is included in the CH3 region of A and a knob structure is included in the CH3 region of C.
- a hole structure is included in the CH3 region of B and a knob structure is included in the CH3 region of D.
- the antigen-binding site, antigen, hinge, linker and Fc are as described above.
- the fusion protein includes polypeptides of structural formulas (I), (II), (III) and (IV), wherein X is a heavy chain variable region and additionally includes CH1, and Y is As a light chain variable region, it includes a light chain constant region. In addition, it binds to the CH1 structure and Cys in the light chain variable region to form a Fab structure.
- n is 0, CH1 is directly bonded to the hinge, m is 1, and L2 includes a peptide linker.
- a and C combine to form a first Fc domain
- B and D combine to form a second Fc domain.
- X and Y combine to form a variable region of Pertuzumab
- VD1 and VD2 combine to form a variable region of Trastuzumab.
- the antigen-binding site, antigen, hinge, linker and Fc are as described above.
- Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the fusion protein as an active ingredient.
- the cancer is gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, skin cancer, bone cancer, multiple myeloma, glioma, ovarian cancer, pancreatic cancer, cervical cancer, thyroid cancer, laryngeal cancer, acute myelogenous leukemia, chronic myelogenous leukemia, acute It may be any one selected from the group consisting of lymphoblastic leukemia, chronic lymphocytic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland cancer, and lymphoma.
- Another aspect of the present invention provides a polynucleotide encoding a polypeptide of formula (I), (II), (III) and/or (IV) above.
- Another aspect of the present invention provides a polynucleotide encoding a polypeptide of formula (I'), (II'), (III) and/or (IV) above.
- the polynucleotide may additionally include a nucleic acid encoding a signal sequence or a leader sequence.
- signal sequence refers to a signal peptide that directs the secretion of a target protein.
- the signal peptide is cleaved after translation in the host cell.
- the signal sequence is an amino acid sequence that initiates movement of a protein through an endoplasmic reticulum (ER) membrane.
- the vector may include a polynucleotide encoding a polypeptide of structure (I), (II), (III) and/or (IV).
- the vector may include a polynucleotide encoding a polypeptide of formula (I'), (II'), (III) and/or (IV).
- the vector can be introduced into a host cell and recombine and integrate into the host cell genome.
- the vector is understood to be a nucleic acid vehicle comprising a polynucleotide sequence capable of autonomous replication as an episome.
- Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogues thereof.
- examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.
- the vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), Escherichia coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis plasmids derived from spp.
- pUB110, pTP5, etc. yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11, ⁇ ZAP, etc.), animal virus vectors (retrovirus ), adenovirus, vaccinia virus, etc.), insect virus vector (Baculovirus, etc.). Since the expression level and modification of the protein of the vector appear differently depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.
- Host cells of the transformed cells may include, but are not limited to, prokaryotic cells, eukaryotic cells, mammalian cells, plants, insects, fungi, or cells of cellular origin.
- prokaryotic cell Escherichia coli may be used.
- yeast may be used as an example of a eukaryotic cell.
- CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells, or HEK293T cells may be used as the mammalian cells.
- any cell that can be used as a mammalian host cell known to those skilled in the art can be used.
- the sugar chain pattern of the fusion protein eg, sialic acid, fucosylation, glycosylation.
- Another aspect of the present invention i) culturing the transformed cells; and ii) recovering the produced fusion protein, providing a method for producing a fusion protein comprising an antigen binding site, a first Fc domain or variant thereof, and a second Fc domain or variant thereof.
- Another aspect of the present invention provides a pharmaceutical composition comprising the fusion protein as an active ingredient.
- the pharmaceutical composition may be used to prevent or treat cancer, for example, gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, gallbladder cancer, bladder cancer, kidney cancer, esophageal cancer, skin cancer, rectal cancer, osteosarcoma, multiple myeloma, glioma, and ovarian cancer.
- cancer for example, gastric cancer, liver cancer, lung cancer, colon cancer, breast cancer, prostate cancer, gallbladder cancer, bladder cancer, kidney cancer, esophageal cancer, skin cancer, rectal cancer, osteosarcoma, multiple myeloma, glioma, and ovarian cancer.
- pancreatic cancer cervical cancer, endometrial cancer, thyroid cancer, laryngeal cancer, testicular cancer, mesothelioma, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, brain tumor, neuroblastoma, retinoblastoma, head and neck cancer, salivary gland It can be used for the prevention or treatment of cancer, which is any one selected from the group consisting of cancer and lymphoma.
- the term “treatment” may be used to include both therapeutic treatment and prophylactic treatment. At this time, prevention may be used in the sense of alleviating or reducing the pathological condition or disease of the subject.
- the term “treatment” includes any form of administration or application to treat a disease in a mammal, including humans. The term also includes inhibiting or slowing the progression of a disease; restoring or repairing a damaged or missing function, thereby partially or completely alleviating the disease; or stimulate inefficient processes; It includes the meaning of alleviating serious illness.
- terapéuticaally effective amount or “pharmaceutically effective amount” is an amount of a compound or composition effective for preventing or treating a target disease, sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment It means an amount that does not cause side effects.
- the level of the effective amount is the patient's state of health, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and It may be determined according to other factors well known in the medical field.
- a therapeutically effective amount refers to an amount of a drug effective to treat cancer.
- the pharmaceutical composition may further include a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier may be any carrier as long as it is a non-toxic material suitable for delivery to a patient. Distilled water, alcohol, fats, waxes and inert solids may be included as carriers. A pharmacologically acceptable adjuvant (buffer, dispersant) may also be included in the pharmaceutical composition.
- the preferred dosage of the pharmaceutical composition is in the range of 0.01 ⁇ g/kg to 10 g/kg, or 0.01 mg/kg to 1 g/kg per day depending on the condition, weight, sex, age, severity of the patient, and route of administration of the patient. can be Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the present invention in any respect.
- compositions are mammals including dogs, cats, humans, etc., and humans are particularly preferred.
- pharmaceutical composition of the present application may further include any compound or natural extract known to have a tumor treatment effect.
- Another aspect of the present invention provides a method for treating or preventing cancer comprising administering to a subject a fusion protein comprising an antigen binding site, a first Fc domain or a variant thereof, and a second Fc domain or variant thereof. do.
- the subject may be a subject suffering from cancer.
- the subject may be a mammal, preferably a human.
- the administration route, dosage and frequency of administration of the fusion protein can be administered to the subject in various ways and amounts depending on the condition of the patient and the presence or absence of side effects, and the optimal administration method, dosage and frequency of administration can be determined by those skilled in the art. range can be selected.
- the fusion protein may be administered in combination with other drugs or physiologically active substances known to have therapeutic effects on the disease to be treated, or formulated in combination with other drugs.
- Natural human immunoglobulin G is composed of two fragment antigen-binding (Fab) regions and one fragment crystallizable (Fc) region (FIG. 1a). Human IgG binds monovalently or bivalently to target antigens and, in some cases, has 0.5 to 1 Fc region per target antigen (FIGS. 1c, 1d, and 1e).
- An object of the present invention is to improve effector function by increasing the amount of Fc region present per antigen while having a molecular weight similar to that of an antibody and having a homogeneous composition. Therefore, a novel antibody structure having two Fc regions similar to the molecular weight of human IgG antibody in nature (about 150 kDa) was devised (Fig. 1b), and this form binds to cancer cell surface antigen compared to existing antibodies, It has a maximum of 4 times the Fc region (Fig. 1b, Fig. 1f).
- Trastuzumab was used as a template to implement the aforementioned novel antibody construct (FIG. 2a).
- an artificial disulfide bond was introduced by substituting a specific amino acid with cysteine (FIGS. 2b, 2c, and 2d).
- the position of the amino acid constituting the antibody follows the Kabat numbering rules.
- Knob-into-hole mutation technology was applied to the Fc domain (SEQ ID NO: 3) of human immunoglobulin G1 (Immunoglobulin G1; IgG1) to , Knob mutation-bearing Fc (S354C, T366W; SEQ ID NO: 4) and Hole mutation-bearing Fc (Y349C, T366S, L368A, Y407V; SEQ ID NO: 5) polypeptides were designed.
- the purified product was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) (FIGS. 4 and 5a to 5d).
- SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
- SEC size exclusion chromatography
- P01 is an expression vector composed of sequences corresponding to Fc-Hole (SEQ ID NO: 7), PerH-G44C-Knob (SEQ ID NO: 29), and PerL-Q100C-Knob (SEQ ID NO: 30), EXPICHO-S TM , Gibco, A29127) was co-transfected (Co-transfection), and purification and analysis were performed in the manner described in Example 1.
- Papain digestion of H01 and P01 was performed.
- Papain (Sigma, P3125) was used after being diluted to 0.1 mg/mL in digestion buffer (20 mM EDTA + 10 mM Cys-HCl in PBS pH 7.4). 200 ⁇ g of H01 and P01 were digested at 37° C. for 2 hours, followed by SDS-PAGE.
- an abnormal (Fc) 2 at about 100 kDa was not confirmed (FIG. 8c, 8d).
- Table 18 shows the nucleotide sequences encoding wtFc of HO1wt, TraH-G44C-wtFc, and TraL-Q100C-wtFc.
- Table 20 shows the polypeptide sequences constituting H01Fv1 to H01Fv7.
- Table 21 shows the nucleotide sequences encoding the polypeptides constituting H01Fv1 to H01Fv7.
- An expression vector composed of sequences corresponding to Fc-Hole-RF (SEQ ID NO: 789), H01Fv1-HC (SEQ ID NO: 790), and H01Fv1-LC (SEQ ID NO: 791) was prepared by EXPICHO-S TM ; Gibco, A29127 ), when co-transfected, H01Fv1 is formed (FIG. 10, Table 20, Table 21). Then, it was purified through affinity chromatography using MABSELECT TM PrismA (Cytiva, 17549853).
- the Fc-Hole (SEQ ID NO: 7) polypeptide can form an Fc-Hole/Fc-Hole dimer, and to prevent this, H435R and Y436F mutations are induced in the Fc-Hole (SEQ ID NO: 7) polypeptide sequence, -RF (SEQ ID NO: 789) polypeptide was prepared (FIG. 10, Table 20, Table 21). This prevents binding to Protein A resin and removes the mispaired Fc-Hole/Fc-Hole dimer (Figs. 10 and 11). As a result of SDS-PAGE analysis, the main product was confirmed near about 130 kDa under non-reducing conditions (NR) (FIG. 11), and monomer purity was confirmed through SEC (FIG. 12).
- H01Fv3 mostly exists in the form of about 65 kDa in an unpaired form, and the fully assembled monomer purity was confirmed to be 14.66% (FIGS. 11 and 12c). It was confirmed that H01Fv1, H01Fv2, H01Fv4, H01Fv5, H01Fv6, and H01Fv7 had monomer purity of 71.24%, 61.25%, 68.55%, 73.05%, 67.73%, and 79.33%, respectively (FIG. 12).
- H01Fv1, H01Fv2, H01Fv4, H01Fv5, H01Fv6, and H01Fv7 were analyzed using Bio-Layer Interferometry (BLI) analysis equipment Octet Red96e (Sartorius) (FIG. 13).
- BBI Bio-Layer Interferometry
- H01Fv1, H01Fv2, H01Fv4, H01Fv5, H01Fv6, and H01Fv7 were analyzed using Bio-Layer Interferometry (BLI) analysis equipment Octet Red96e (Sartorius) (FIG. 13).
- Human Her2 recombinant protein R&D systems, 1129-ER
- H01Fv5H01Fv6, and H01Fv7 are calculated (FIG. 13, Table 22).
- the T m1 values were 68, 68, 66, and 66 ° C for Trastuzumab, Pertuzumab, H01, and P01, respectively, and the T m2 values were 81, 79, 83, and 83 ° C, respectively. It was confirmed (FIG. 14, Table 23).
- the binding shift values of H01 + P01 bound to the HER2-loaded sensor and the binding shift values of Trastuzumab + Pertuzumab were 1.477 nm (y-axis value at 4140 s - y-axis value at 1260 s), 0.923 nm (y-axis value at 4140 s), respectively. axis value - y axis value of 1260 s).
- the binding shift value increases, so it was confirmed that when H01 and P01 were bound to the same amount of HER2, a greater amount of antibody was bound than when Trastuzumab and Pertuzumab were bound ( Figure 15, Table 24).
- P01DE4 is Fc-Hole-S239D-I332E (Table 26, Table 27, and SEQ ID NO: 42), PerH-G44C-Knob-S239D-I332E (Table 26, Table 27, and SEQ ID NO: 45), PerL-Q100C-Knob- A vector capable of expressing a polypeptide corresponding to S239D-I332E (Table 26, Table 27, and SEQ ID NO: 46) was co-transfected with EXPICHO-S TM (Gibco, A29127) manufactured.
- K a and K d values were calculated through a 1:1 binding model in Octet analysis software (Sartorius), and based on this, the equilibrium dissociation constant (K D ) value was determined (FIGS. 21a to 21h, 22a to 22a). 22h, FIGS. 23A-23H, Table 29).
- Standard quantitative samples of H01, P01 Trastuzumab, and Pertuzumab were prepared and used for quantification, and the concentration of the analyte at each time was quantified through a standard curve generated from naive rat serum containing each concentration.
- Intravenous administration the half-lives of H01, P01, Trastuzumab, and Pertuzumab were confirmed to be about 11.8 days, 14.2 days, 7.3 days, and 11.6 days, respectively (FIGS. 24a and 24b, Table 30). It was confirmed that H01 and P01, the novel engineered constructs, had PK parameters similar to those of the humanized antibodies Trastuzumab and Pertuzumab.
- a biparatopic antibody structure HP501 was designed in which the V domains of Trastuzumab and Pertuzumab were connected by a linker (FIG. 25).
- the length of the linker connecting the V domains is changed, and at the same time, a Cys substitution mutation that can form a disulfide bond with the V domain is introduced to improve protein properties. 16 constructs were designed (FIG. 26, Table 31). According to the Kabat numbering standard, mutations were introduced only at position 44 for the heavy chain and position 100 for the light chain (Table 31).
- VH linker with 6 amino acid residues is VH-S-Linker
- VH linker with 13 amino acid residues is VH-L-Linker
- VL linker with 6 amino acid residues is VL-S-Linker
- 13 amino acid residues The VL linker has been named VL-L-Linker.
- HP501 is an expression vector composed of sequences corresponding to Fc-Hole (SEQ ID NO: 7), TH-S-PH-Knob (SEQ ID NO: 51), and TL-S-PL-Knob (SEQ ID NO: 59) Expicho-S ( EXPICHO-S TM ; Gibco, A29127) was co-transfected (Tables 31, 32, 33, 34), and purified and analyzed in the manner described in Example 1. Expression and purification analysis were also performed for HP502 - HP516 in the same manner as described above, and the configuration of the expression vector is shown in detail in Tables 32 and 34.
- the binding constants of the D2 and D4 regions of the HER2 protein of HP501, HP502, HP503, HP504, HP505, HP506, HP507, HP508, HP509, HP510, HP511, HP512, HP513, HP514, HP515, and HP516 were analyzed by Octet Red96e (Sartorius). was measured using For analysis of binding constants of 16 antibodies to the D2 region, human Her2 recombinant protein (R&D systems, 1129-ER) was loaded onto the Anti-Penta-HIS (HIS1K) biosensor (Sartorius, 18-5120), followed by D4 region. Blocking was induced using 100 nM Trastuzumab targeting .
- binding reactions 300 seconds and dissociation reactions (600 seconds) were induced for 16 types of antibodies at a concentration of 100 nM, and based on this, the affinity for the D2 region was calculated (Table 36).
- human Her2 recombinant protein R&D systems, 1129-ER
- HIS1K Anti-Penta-HIS biosensor
- binding reactions 300 seconds and dissociation reactions (600 seconds) were induced for 16 types of antibodies at a concentration of 100 nM, and based on this, the affinity for the D4 region was calculated (Table 36).
- the binding constants of HP507, HP511, and HP515 to D2 were 2.285, 3.267, and 2.012 nM, respectively, showing excellent binding ability to the D2 region compared to other clones (Table 36).
- HP503 has a binding constant of 8.098 nM to the D2 region and shows a relatively low binding capacity to the D2 region compared to HP507, HP511, and HP515. , It was confirmed that strong binding capacity was shown at 0.227 nM (Table 36).
- HP503, HP507, HP511, and HP515 were measured using Octet Red96e (Sartorius) in the same manner as in Example 10 (FIGS. 29a to 29d, Table 38).
- HP503, HP507, HP511, and HP515 had excellent binding ability to Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32A, 131R), and Fc ⁇ RIIIA (CD16A, 176V) compared to human IgG1, Trastuzumab, Pertuzumab, and Margetuximab (FIG. 21a to 21h, 22a to 22h, 23a to 23h, 29a to 29d, Table 29, Table 38).
- the binding constant analysis of HP503, HP507, HP511 and HP515 to the neonatal Fc Receptor (FcRn) was measured using Octet Red96e (Sartorius).
- Anti-Human Fab-CH1 2nd Generation (FAB2G) biosensor (Sartorius, 18-5126) was loaded with HP503, HP507, HP511, HP515, Human IgG1 (Bio X cell, BE0297), Trastuzumab, Pertuzumab, Margetuximab, and binding time and dissociation time was analyzed to be 120 seconds, respectively (Figs. 30a to 30b, Table 39).
- Kinetics Buffer (Sartorius, 18-1105) was used adjusted to pH 6.0.
- BT474 HER2 3+; High breast cancer cell line
- NCI-N87 HER2 3+; High gastric cancer cell line
- Cells were diluted and dispensed in a cell culture medium to 10,000 cells per well in a 96-well plate.
- Cells, antibodies, and human serum were reacted at a volume ratio of 1:1:1, respectively.
- Cell Titer Glo-Reagent Promega, G9243 previously melted at 4°C was dispensed into each well in the same volume of the mixed culture medium, and then cells were plated on a plate shaker (Allsheng, MX100-4A) at a rotation speed of 500 rpm for 2 minutes. lysis was induced. After incubation for 10 minutes at room temperature for stabilization of the luminescence signal, analysis was performed using a plate reader (Envision; PerkinElmer, 2105-0010) (FIGS. 31a to 31b). Complement-dependent cytotoxicity (CDC activity) was calculated as follows.
- PBMCs Peripheral Blood Mononuclear Cells
- H01 In NCI-N87 (HER2 3+; High) and MDA-MB-453 (HER2 2+; Mid) cancer cell lines, H01 exhibited superior cytotoxicity than Trastuzumab at a lower concentration (FIGS. 32a, 32b). As a result of cytotoxicity analysis performed on SNU-601 (HER2 1+; Low) and SNU-5 (HER2 1+; Low) cancer cell lines, H01 exhibited superior cytotoxicity compared to Trastuzumab (FIGS. 32c and 32d).
- SNU-5 gastric cancer cell line-derived xenograft model was performed using 6-week-old female SCID mice (CB-17/NcrKoat-Prkdc scid , Koatech) ( Figure 33a).
- the SNU-5 cancer cell line was diluted in PBS at 1 x 10 7 cells/100 ⁇ L, mixed 1:1 with MATRIGEL ® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, 354230), and 100 ⁇ L of the mixture was transplanted subcutaneously in the right flank. and observed tumor growth.
- Regroup was performed so that the average tumor volume was about 107 mm 3 , PBS (Vehicle), 5 mg/kg H01, 5 mg/kg H01 + 5 mg/kg P01, 5 mg/kg HP507, 5 mg/kg Trastuzumab, 5 mg/kg Trastuzumab + 5 mg/kg Pertuzumab was administered intravenously (IV) once a week for a total of 6 weeks (FIG. 33a).
- IV intravenously
- Regrouping was performed so that the average tumor volume was approximately 122 mm 3 , and 50 mg/kg intravenous immunoglobulin (IVIG; LIV-r, SK Plasma) was administered to all mice twice a week for 6 weeks (FIG. 33b).
- IVIG intravenous immunoglobulin
- PBS Vehicle
- 1 mg/kg Trastuzumab, 1 mg/kg Pertuzumab 0.5 mg/kg Trastuzumab + 0.5 mg/kg Pertuzumab, 1 mg/kg H01, 1 mg/kg P01, 0.5 mg/kg H01 + 0.5 mg/kg P01 was administered intraperitoneally (IP) twice a week for a total of 6 weeks (FIG. 33B).
- NCI-N87 gastric cancer cell line-derived xenograft model was performed using 6-week-old female SCID mice (CB-17/NcrKoat-Prkdc scid , Koatech).
- the NCI-N87 cancer cell line was diluted in PBS at 5 x 10 6 cells/100 ⁇ L, mixed 1:1 with MATRIGEL ® Growth Factor Reduced (GFR) Basement Membrane Matrix (Corning, 354230), and 100 ⁇ L of the mixture was transplanted subcutaneously in the right flank. and observed tumor growth.
- Regroup was performed so that the average tumor volume was approximately 146 mm 3 , and PBS (Vehicle), 0.2 mg/kg H01, 5 mg/kg H01, 0.2 mg/kg Trastuzumab, and 5 mg/kg Trastuzumab were intraperitoneally administered (IP). It was administered twice a week for a total of 6 weeks (FIG. 35). Since antibodies do not exist in the body of SCID mice, 50 mg/kg intravenous immunoglobulin (IVIG; LIV-r, SK Plasma) was administered to all mice twice a week for 6 weeks to mimic the actual human blood environment (Fig. 35). As a result of the analysis, H01 exhibited superior anticancer activity compared to Trastuzumab at doses of 5 mg/kg and 0.2 mg/kg (FIG. 35).
- IVIG intravenous immunoglobulin
- Nucleotides (SEQ ID NO: 566, Table 40) encoding human HER2 protein (SEQ ID NO: 567, Table 40) were cloned into a protein expression vector (ORIGENE, PS100020) containing a Neomycin resistance gene to obtain human HER2 expression vector pCMV6-AC-hHER2 was built (FIG. 36, Table 40).
- the pCMV6-AC-hHER2 human HER2 expression vector was transfected into CT26 mouse colon cancer cells using lipofectamine 2000 transfection reagent (Invitrogen, 11668-019). Only cells transfected with the pCMV6-AC-hHER2 human HER2 expression vector were selected in a culture medium containing 1 mg/mL G418 (Invivogen, ant-gn-5) for 14 days. Using the SH800S Cell Sorter (SONY), the top 3% population of the expression level was sorted into a 96-well plate (ThermoFisher, 167008) so that one cell per well was entered.
- GPM01 is an expression vector composed of sequences corresponding to Fc-Hole (SEQ ID NO: 7), GPM01 HC (SEQ ID NO: 67), and GPM01 LC (SEQ ID NO: 68) in EXPICHO-S TM (Gibco, A29127) Co-transfection (Co-transfection) was performed (Fig. 41a, Table 44), purification and analysis were performed in the manner described in Example 1. Expression and purification analysis of GPM02, GPM04, GPB01, GPB03, GPB04, and GPB06 was performed in the same manner as described above (FIG. 41a to FIG.
- Table 46 below shows the polypeptide sequences of the modified antibody heavy chain variable region (VH) and light chain variable region (VL) targeting GPC-3.
- Table 47 below shows the nucleotide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of modified antibodies targeting GPC-3.
- Table 48 below shows the modified antibody heavy and light chain CDR sequences targeting GPC-3.
- Table 52 below shows the polypeptide sequences of the modified antibody heavy chain variable region (VH) and light chain variable region (VL) targeting EphA2.
- Table 53 below shows the nucleotide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of modified antibodies targeting EphA2.
- PC-3 prostate cancer cell line was used for the analysis of EphA2 signal transduction inhibition ability. Lysates of PC-3 cancer cell lines treated with each antibody at a concentration of 50 nM for 30 minutes were analyzed by western blot. 1C1 human EphA2 target antibody was used as a positive control and was prepared based on a sequence published in the literature (Kinch et al., US 20090304721 A1).
- Table 60 shows the modified antibody heavy and light chain CDR sequences targeting MET.
- EGM01 is an expression vector composed of sequences corresponding to Fc-Hole (SEQ ID NO: 7), EGM01 HC (SEQ ID NO: 590), and EGF01 LC (SEQ ID NO: 591) in EXPICHO-S TM (Gibco, A29127) Co-transfection (Co-transfection) was carried out (Fig. 41a, Table 62), and purification and analysis were performed in the manner described in Example 1.
- EGM02, EGM03, EGM04, EGM05, and EGM06 were also subjected to expression and purification analysis in the same manner as described above (Table 62).
- Binding constants of EGM01 to EGM05 to the EGFR protein were measured using an Octet Red96e (Sartorius) analyzer.
- Anti-Human Fab-CH1 2nd Generation (FAB2G) biosensor (Sartorius, 18-5125) was loaded with antibody to analyze the binding constant of the antibody, and human EGFR recombinant protein (Sino Biologicals, 10692-H08H) was bound at various concentrations A reaction (300 sec) and a dissociation reaction (600 sec) were induced (FIG. 49). Based on this, the affinity for EGFR was calculated (FIG. 49, Table 67). Table 67 below analyzes binding constants of modified antibodies targeting EGFR.
- GPM01 is an expression vector composed of sequences corresponding to Fc-Hole (SEQ ID NO: 7), 33-1 HC (SEQ ID NO: 636), and 33-1 LC (SEQ ID NO: 637), EXPICHO-S TM ; Gibco , A29127) was co-transfected (Fig. 41a, Table 64), and purification and analysis were performed in the manner described in Example 1. 33-2, 33-3, 33-4, 33-5, 33-6, and 33-7 were also subjected to expression and purification analysis in the same manner as described above (FIGS. 41a to 41b, Tables 68 and 69) .
- 33-1, 33-2, and 33-3 bind monovalently to different antigen epitopes and are composed of two Fc domains (FIG. 41a).
- 33-4, 33-5, 33-6, and 33-7 are structures in which the variable region of the CD33 antibody is connected with a polypeptide linker (SEQ ID NO: 48, SEQ ID NO: 50), biparatopic binding to CD33, and composed of two Fc domains. This is the constructed structure (Fig. 41b).
- Table 70 below shows the heavy chain variable region (VH) and light chain variable region (VL) polypeptide sequences of modified antibodies targeting CD33.
- Table 71 below shows the nucleotide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of modified antibodies targeting CD33.
- Table 72 below shows the modified antibody heavy and light chain CDR sequences targeting CD33.
- CD33 protein binding constants of 33-1, 33-2, 33-3, 33-4, 33-5, 33-6, and 33-7 were measured using an Octet Red96e (Sartorius) analyzer.
- human CD33 recombinant protein (Sino Biologicals, 12238-H08H) was loaded onto the Anti-Penta-HIS (HIS1K) biosensor (Sartorius, 18-5120), and then the 7 antibodies were Association reactions (600 seconds) and dissociation reactions (1,200 seconds) were induced at various concentrations, and based on these, the affinity for CD33 was calculated (FIG. 50, Table 73). Table 73 below analyzes binding constants of modified antibodies targeting CD33.
- Example 22 Design and manufacture and analysis of antibody constructs targeting CEACAM5
- CEA01 is an expression vector composed of sequences corresponding to Fc-Hole (SEQ ID NO: 7), CEA01 HC (SEQ ID NO: 590), and CEA01 LC (SEQ ID NO: 591) in EXPICHO-S TM (Gibco, A29127) Co-transfection (Co-transfection) was performed (Fig. 41a, Table 74), purification and analysis were performed in the manner described in Example 1.
- CEA02, CEA03, and CEA04 were also subjected to expression and purification analysis in the same manner as described above (Table 74).
- Table 75 below shows the nucleotide sequences of modified antibody heavy and light chains targeting CEACAM5.
- Table 76 shows the heavy chain variable region (VH) and light chain variable region (VL) polypeptide sequences of modified antibodies targeting CEACAM5.
- Table 77 shows the nucleotide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of modified antibodies targeting CEACAM5.
- Table 78 shows the heavy and light chain CDR sequences of modified antibodies targeting CEACAM5.
- human CEACAM5 recombinant protein (Sino Biologicals, 11077-H08H) was loaded onto the Anti-Penta-HIS (HIS1K) biosensor (Sartorius, 18-5120), and the antibodies were combined at various concentrations (600 seconds). and dissociation reactions (1,200 sec) were induced, and based on this, the affinity for human CEACAM5 was calculated (FIG. 51, Table 79). Table 79 below analyzes binding constants of modified antibodies targeting human CEACAM5.
- Example 23 Design and manufacture and analysis of antibody constructs targeting TROP2 or Mesothelin or LIV-1
- Table 80 shows the light and heavy chain variant polypeptide sequences of antibody T01 that specifically binds to the TROP2 protein.
- T01 an expression vector composed of sequences corresponding to Fc-Hole (SEQ ID NO: 7), T01 HC (SEQ ID NO: 753), and T01 LC (SEQ ID NO: 754) was cotransformed into ExpiCHO-S TM (Gibco, A29127) (Co -transfection) (FIG. 41a, Table 80), and purification and analysis were performed in the manner described in Example 1.
- Table 80 shows the light and heavy chain variant polypeptide sequences of antibody MSM01 that specifically binds to mesothelin protein.
- MSM01 is an expression vector composed of sequences corresponding to Fc-Hole (SEQ ID NO: 7), MSM01 HC (SEQ ID NO: 755), and MSM01 LC (SEQ ID NO: 756) co-transformed into EXPICHO-S TM (Gibco, A29127) (Co -transfection) (FIG. 41a, Table 80), and purification and analysis were performed in the manner described in Example 1.
- Table 80 shows the light and heavy chain variant polypeptide sequences of the antibody LIM01 that specifically binds to the LIV-1 protein.
- LIM01 an expression vector composed of sequences corresponding to Fc-Hole (SEQ ID NO: 7), LIM01 HC (SEQ ID NO: 757), and LIM01 LC (SEQ ID NO: 758) was cotransformed into EXPICHO-S TM (Gibco, A29127) (Co -transfection) (FIG. 41a, Table 80), and purification and analysis were performed in the manner described in Example 1.
- Table 81 shows the modified antibody heavy and light chain nucleotide sequences targeting TROP2 or Mesothelin or LIV-1.
- Table 82 shows the heavy chain variable region (VH) and light chain variable region (VL) polypeptide sequences of modified antibodies targeting TROP2 or Mesothelin or LIV-1.
- Table 83 shows the nucleotide sequences of the heavy chain variable region (VH) and light chain variable region (VL) of modified antibodies targeting TROP2 or Mesothelin or LIV-1.
- Table 84 shows the heavy and light chain CDR sequences of modified antibodies targeting TROP2 or Mesothelin or LIV-1.
- human TROP2 recombinant protein (Sino Biologicals, 10428-H08H) or human Mesothelin recombinant protein (Sino Biologicals, 13128-H08H) or human LIV-1 recombinant protein (Acro biosystems, LV1-H5223) was Anti-Penta- It was loaded onto the HIS (HIS1K) biosensor (Sartorius, 18-5120), and binding and dissociation reactions were induced at various concentrations, and based on this, the affinity of each antibody for the human antigen was calculated (Fig. 52, Table 85). Table 85 below analyzes binding constants of modified antibodies targeting TROP2 or Mesothelin or LIV-1.
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Abstract
Description
Claims (23)
- (a) 적어도 하나의 complementarity-determining region (CDR) 서열을 포함하는 제1 폴리펩티드와 적어도 하나의 complementarity-determining region (CDR) 서열을 포함하는 제2 폴리펩티드로 이루어지고, 상기 제1 폴리펩티드와 상기 제2 폴리펩티드가 이량체 (dimer)를 형성하고, 타겟 항원에 특이적으로 결합할 수 있는 항원 결합 부위,(b) 두 개의 폴리펩티드 서열로 이루어진 이량체로서, 이중 하나의 폴리펩티드 서열이 상기 항원 결합 부위의 제1 폴리펩티드에 결합된 제1 Fc 도메인 또는 이의 변이체, 및(c) 두 개의 폴리펩티드 서열로 이루어진 이량체로서, 이중 하나의 폴리펩티드 서열이 상기 항원 결합 부위의 제2 폴리펩티드에 결합된 제2 Fc 도메인 또는 이의 변이체를 포함하는 융합단백질.
- 제1항에 있어서,상기 항원 결합 부위의 제1 폴리펩티드는 항체 중쇄의 CDR1, CDR2, 및 CDR3를 포함하고, 상기 항원 결합 부위의 제2 폴리펩티드는 항체 경쇄의 CDR1, CDR2, 및 CDR3를 포함하는 것인, 융합단백질.
- 제2항에 있어서,상기 항원 결합 부위의 제1 폴리펩티드는 항체 중쇄의 CH1 영역을 더 포함하고, 및/또는 상기 항원 결합 부위의 제2 폴리펩티드는 항체 경쇄의 불변 영역을 더 포함하는 것인, 융합단백질.
- 제1항에 있어서,상기 항원 결합 부위는 세포 표면에 발현되는 단백질에 특이적으로 결합하는 것인, 융합단백질.
- 제1항에 있어서,상기 항원 결합 부위는 암 항원에 특이적으로 결합하는 것인, 융합단백질.
- 제1항에 있어서,상기 융합단백질은, 동일한 항원 결합 부위를 가지는 통상적 구조의 IgG 기반 항체 대비 향상된 종양억제능을 유도하는 것인, 융합단백질.
- 제1항에 있어서,상기 항원 결합 부위는 PD-L1, EGFR, EGFRvIII, BCMA, CD22, CD25, CD30, CD33, CD37, CD38, CD52, CD56, CD123, c-Met, DLL3, DR4, DR5, GD2, Nectin-4, RANKL, SLAMF7, Trop-2, LIV-1, Claudin 18.2, IL13α2, CD3, HER2, HER3, FGFR2, FGFR3, GPC3, ROR1, Folα, CD20, CD19, CTLA-4, VEGFR, NCAM1, ICAM-1, ICAM-2, CEACAM5, CEACAM6, Carcinoembryonic antigen (CEA), CA-125, Alphafetoprotein (AFP), MUC-1, MUC-16, PSMA, PSCA, Epithelial tumor antigen (ETA), Melanoma-associated antigen (MAGE), Immature laminin receptor, TAG-72, HPV E6/E7, BING-4, Calcium-activated chloride channel 2, Cyclin-B1, 9D7, Ep-CAM, EphA2, EphA3, Mesothelin, SAP-1, Survivin 및 바이러스 유래 항원으로 이루어진 그룹에서 선택되는 어느 하나에 특이적으로 결합하는 것인, 융합단백질.
- 제1항에 있어서,상기 제1 Fc 도메인 및 상기 제2 Fc 도메인은 각각 야생형 Fc 도메인 또는 Fc 도메인 변이체인 것인, 융합단백질.
- 제8항에 있어서,상기 Fc 영역은 IgG, IgA, IgE, IgD 또는 IgM의 Fc 영역 또는 이의 변이체인 것인, 융합단백질.
- 제8항에 있어서,상기 제1 Fc 도메인 변이체 및 제2 Fc 도메인 변이체는 각각 독립적으로 Fc 이종이량체(heterodimeric Fc) 형성을 촉진하는 놉 구조 또는 홀 구조를 포함하는 것인; 및/또는상기 제1 Fc 도메인 변이체 및 제2 Fc 도메인 변이체는 각각 독립적으로 정전기 조향(electrostatic steering) 기작에 의한 이종이량체 형성을 촉진하는 변이체를 포함하는 것인, 융합단백질.
- 제1항에 있어서,상기 융합단백질은 하기 구조식 (I), (II), (III) 및 (IV)의 폴리펩티드를 포함하는 것인, 융합단백질:N'-X-(L1)n-A-C' (I);N'-Y-(L2)m-B-C' (II);N'-C-C' (III); 및N'-D-C' (IV)이때, 상기 구조식 (I), (II), (III) 및 (IV)에 있어서,상기 N'은 각 폴리펩티드의 N-말단이고,상기 C'은 각 폴리펩티드의 C-말단이며,상기 - 는 결합을 의미하고,상기 A, B, C 및 D는 각각 면역글로불린의 CH2 및 CH3 영역을 포함하여, 임의적으로 CH4 및/또는 힌지 서열을 더 포함하는 Fc 도메인의 단량체 폴리펩티드 서열이며, 상기 A는 C 또는 D중 하나와 함께 이량체를 형성하여 상기 제1 Fc 도메인 (b)를 형성하고, 상기 B는 C 또는 D중 남은 하나와 함께 이량체를 형성하여 상기 제2 Fc 도메인 (c)를 형성하며;L1 및 L2는각각 펩티드 링커이며,상기 n 및 m은 각각 독립적으로 0 또는 1이며,상기 X는 상기 항원 결합 부위의 제 1 폴리펩티드 서열로서, 제 1 항원에 특이적으로 결합하는 항체의 중쇄 CDR1, CDR2, CDR3 서열을 포함하는 서열이거나 혹은 제 1 항원에 특이적으로 결합하는 항체의 중쇄 가변영역을 포함하고;상기 Y는 상기 항원 결합 부위의 제 2 폴리펩티드 서열로서, 제 1 항원에 특이적으로 결합하는 항체의 경쇄 CDR1, CDR2, CDR3 서열을 포함하는 서열이거나 혹은 제 1 항원에 특이적으로 결합하는 항체의 경쇄 가변영역을 포함하며;상기 X 및 Y는 결합하여 항원에 특이적으로 결합하는 상기 항원 결합 부위 (a)를 형성한다.
- 제11항에 있어서,상기 구조식 (I)의 X는 추가적으로 중쇄 CH1 영역을 포함하고, 및/또는상기 구조식 (II)의 Y는 추가적으로 경쇄 불변영역을 포함하는 것인, 융합단백질.
- 제1항에 있어서,상기 융합단백질은 하기 구조식 (I'), (II'), (III) 및 (IV)의 폴리펩티드를 포함하는 것인, 융합단백질:N'-VD1-(L3)p-X-(L1)n-A-C' (I');N'-VD2-(L4)q-Y-(L2)m-B-C' (II');N'-C-C' (III); 및N'-D-C' (IV)이때, 상기 구조식 (I'), (II'), (III) 및 (IV)에 있어서,상기 N'은 각 폴리펩티드의 N-말단이고,상기 C'은 각 폴리펩티드의 C-말단이며,상기 - 는 결합을 의미하고,상기 A, B, C 및 D는 각각 면역글로불린의 CH2 및 CH3 영역을 포함하여, 임의적으로 CH4 및/또는 힌지 서열을 더 포함하는 Fc 도메인의 단량체 폴리펩티드 서열이며, 상기 A는 C 또는 D중 하나와 함께 이량체를 형성하여 상기 제1 Fc 도메인 (b)를 형성하고, 상기 B는 C 또는 D중 남은 하나와 함께 이량체를 형성하여 상기 제2 Fc 도메인 (c)를 형성하며;L1, L2, L3 및 L4는 펩티드 링커이며,상기 n, m, p 및 q는 각각 0 또는 1이며,상기 VD1은 항원에 특이적으로 결합하는 항체의 중쇄 혹은 경쇄 가변영역 혹은 항체 중쇄 혹은 경쇄 CDR1, CDR2, 및 CDR3으로 구성되고;상기 VD2는 항원에 특이적으로 결합하는 항체의 경쇄 혹은 중쇄 가변영역 혹은 항체 중쇄 혹은 경쇄 CDR1, CDR2, 및 CDR3으로 구성되며;상기 VD1과 VD2는 결합하여 제2 항원에 특이적으로 결합하는 제2 항체 가변 영역을 형성하며,상기 X는 항원에 특이적으로 결합하는 항체 중쇄 혹은 경쇄 가변영역 혹은 항체 중쇄 혹은 경쇄 CDR1, CDR2, 및 CDR3을 포함하고;상기 Y는 항원에 특이적으로 결합하는 항체 경쇄 혹은 중쇄 가변영역 혹은 항체 중쇄 혹은 경쇄 CDR1, CDR2, 및 CDR3을 포함하며;상기 X 및 Y는 결합하여 제1 항원에 특이적으로 결합하는 제1 항체 가변 영역을 형성하고,상기 VD1-(L3)p-X는 상기 항원결합 부위(a)의 제1 폴리펩티드 서열을 형성하고, 상기 VD2-(L4)q-Y는 상기 항원결합 부위(a)의 제2 폴리펩티드 서열을 형성한다.
- 제13항에 있어서,상기 중쇄 가변영역은 추가적으로 중쇄 CH1 영역을 포함하고,상기 경쇄 가변영역은 추가적으로 경쇄 불변영역을 포함하는 것인, 융합단백질.
- 제11항 또는 제13항에 있어서,상기 Fc 도메인 단량체는 Fc 이종이량체(heterodimeric Fc) 형성을 촉진하는 놉 구조 또는 홀 구조를 포함하는 것인; 또는상기 Fc 도메인 단량체는 정전기 조향(electrostatic steering) 기작에 의한 이종이량체 형성을 촉진하는 변이체를 포함하는 것인, 융합단백질.
- 제12항 또는 제14항에 있어서,상기 X 및 Y의 결합은i) CH1 및 경쇄 불변영역에 존재하는 Cys에 의한 이황화 결합을 통해 이루어 지거나,ii) 중쇄 가변영역 및 경쇄 가변영역에 존재하는 Cys에 의한 이황화 결합을 통해 이루어 지거나,iii) CH1 및 경쇄 불변영역에 존재하는 Cys에 의한 이황화 결합 및 중쇄 가변영역 및 경쇄 가변영역에 존재하는 Cys에 의한 이황화 결합을 통해 이루어 지는 것을 특징으로 하는, 융합단백질.
- 제14항에 있어서,상기 X 및 Y의 결합은 Kabat numbering기준 CH1233와 CL214 사이에 존재하는 이황화 결합 이외에 추가적으로,i) VH105와 VL43 사이에 존재하는 이황화 결합, 또는;ii) VH44와 VL100 사이에 존재하는 이황화 결합, 또는;iii) CH1122과 CL121 사이에 존재하는 이황화 결합을 포함하는 것인, 융합단백질.
- 제1항 내지 제17항 중 어느 한 항의 융합단백질을 유효성분으로 포함하는 암 예방 또는 치료용 약학 조성물.
- 제18항에 있어서,상기 암은 위암, 간암, 폐암, 대장암, 유방암, 전립선암, 담낭암, 방광암, 신장암, 식도암, 피부암, 직장암, 골육종, 다발성골수종, 신경교종, 난소암, 췌장암, 자궁경부암, 자궁내막암, 갑상선암, 후두암, 고환암, 중피종, 급성 골수성 백혈병, 만성 골수성 백혈병, 급성 림프모구성 백혈병, 만성 림프모구성 백혈병, 뇌종양, 신경모세포종, 망막 모세포종, 두경부암, 침샘암 및 림프종으로 구성된 군에서 선택되는 어느 하나인, 약학 조성물.
- 제1항 내지 제17항 중 어느 한 항의 융합단백질을 발현하는 형질전환 세포.
- 제1항에 따른 융합단백질을 암 치료 혹은 암 예방이 필요한 개체에 투여하는 단계를 포함하는 암을 치료 또는 예방하는 방법.
- 암을 치료하기 위한, 제1항에 따른 융합단백질의 용도.
- 암을 치료용 약물을 제조하는데 사용하기 위한, 제1항에 따른 융합단백질의 용도.
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